WO2023222986A1

WO2023222986A1 - Method for preparing glycolipids, glycolipids and uses thereof

Info

Publication number: WO2023222986A1
Application number: PCT/FR2023/050717
Authority: WO
Inventors: Etienne Severac; Magali Remaud-Simeon; David GUIEYSSE; Claire Moulis; Didier Pintori; Vanessa KOMOROWSKI; Chrystel Faure; Fernando Leal Calderon
Original assignee: Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement; Centre National De La Recherche Scientifique (Cnrs); Institut National Des Sciences Appliquees De Toulouse; Universite de Bordeaux; Institut Polytechnique De Bordeaux
Priority date: 2022-05-18
Filing date: 2023-05-17
Publication date: 2023-11-23

Abstract

The present invention relates to a method for preparing at least one glycolipid according to the following formula (I): [Glc]n-xOy-R (I) [Glc]n representing a linear or branched osidic motif comprising n glucosyl units, where n is between 1 and 7, with the condition that when the osidic motif comprises a plurality of glucosyl units, the latter are linked to one another by α-osidic bonds; R representing a fatty acid radical comprising between 4 and 24 carbon atoms, the carbon chain of which is linear or branched, saturated or unsaturated; -xOy- symbolising the attachment of the fatty acid radical to the osidic motif via an ether bond linking a carbon atom Cx of a glucosyl residue of the osidic motif, previously bearing a hydroxyl group, to a carbon atom Cy of the fatty acid radical, previously bearing a hydroxyl group, where Cx is the position of the atom of the glucosyl residue to which the bond is made, Cx representing the carbon atom C1 of the glucosyl residue, Cy being a carbon atom positioned along the carbon chain of the fatty acid radical or at the omega end thereof; the method comprising a step of glucosylating a hydroxylated fatty acid of formula (II): R-yOH (II) -yOH representing a hydroxyl group attached to a carbon atom Cy as defined above; the step comprising bringing the hydroxylated fatty acid of formula (II) into contact with at least one α-transglucosylase of the GH70 family in the presence of saccharose or a saccharose analogue.

Description

PROCESS FOR PREPARING GLYCOLIPIDS, GLYCOLIPIDS AND THEIR USES

The present invention relates to the field of enzymatic preparation of glycolipids. The present invention also relates to glycolipids and their uses.

Glycolipids are compounds comprising a sugar part and a lipid part. Glycolipids can be of natural or biosynthetic origin. Glycolipids have a certain attractiveness due to their amphiphilic character and their solubility properties.

Natural glycolipids are mainly sophorolipids, made up of a sophorose entity (0-1,2-linked glucose disaccharide) and a C16 or C18 fatty acid chain with unsaturation. Another category of natural glycolipids is represented by those of rhamnolipids consisting of one or two rhamnosyl units linked to a fatty tail of 3-(hydroxyalkanoyloxy)-alkanoic acid (mainly C10 fatty acids).

Synthetic glycolipids are mainly carbohydrate esters (glucose, sucrose, maltose) also called sucroesters, in which the sugar part and the lipid part are linked by an ester bond, or else alkyl-(poly-)glucosides also called polyglucosides. alkyl, in which the sugar part and the lipid part are connected by an ether bond.

Currently, glycolipids are classically synthesized via chemical pathways. For example, the most common procedures for synthesizing alkyl-(poly-)glycosides include the Fischer method, the Koenig-Knorr method, or the Schmidt method.

Fischer's method is the most widely used in industry. It allows the production of short-chain alkyl-polyglucosides in a single step. The carbohydrate is solubilized in an excess of alcohol in the presence of an acid catalyst (sulfonic acid, hydrochloric acid) and at elevated temperature (von Rybinski and Hill, Angew. Chem. Int. Ed. 37, 1328-1345, 1998) . Obtaining APGs of larger sizes requires an additional step in which a small alkyl (generally butyl) is exchanged with an alcohol of higher degree, thus making it possible to circumvent the problems of solubility of the reagents. This synthesis method is efficient but the acid catalysts are difficult to recycle. In addition, it is difficult to apply to obtaining di- or tri-glycosylated derivatives due to a competitive alcoholysis reaction of interglycosidic bonds (Koto et al., Simple preparations of alkyl and cycloalkyl a-glycosides of maltose, cellobiose and lactose, Carbohydr. Res. 339, 2415-2424, 2004).

The chemical synthesis of glycolipids of the alkyl-polyglucoside type therefore involves a multitude of reaction steps including carbohydrate protection/deprotection steps; the use of acidic, alkaline or metallic catalysts and large quantities of solvents to be recycled; risks of side reactions due to the reactivity of the catalysts and the drastic synthesis conditions; the formation of both anomers in glycosylation products which are difficult to separate.

The biosynthesis of ether-linked glycolipids is possible enzymatically. The enzymes most studied in this regard are those of the p-glycosidases family. The latter are enzymes without co-factors which naturally hydrolyze saccharide bonds present in polysaccharides to produce mono- or oligosaccharides. In vitro, these enzymes are capable of using a glycosylated donor and catalyzing the transfer of the glycosylated onto a free hydroxyl of an acceptor molecule and thus catalyzing the formation of an ether bond between the acceptor molecule and the glycosylated residue. The synthesis of alkyl-polyglucosides catalyzed by p-glycosidases generally retains a p-configuration bond between the sugars and the alkyl part (P-alkyl-polyglucosides).

This synthesis is possible either by reversion of hydrolysis or by transglycosylation.

In the hydrolysis reversion approach, the glycosylated donors are polysaccharides or carbohydrates such as starch, cellulose, sucrose or glucose. In this case, the thermodynamic equilibrium is rather favorable to hydrolysis, the production yields of alkyl-polyglucosides are therefore generally very modest. For example, several teams working on almond p-glucosidase have obtained production yields lower than 62% with short chain alcohols (ie methylglucoside/xyloside or ethylglucoside/xyloside). The yields with longer chain alcohols (carbon number greater than 6) are even lower and between 1 and 13% (Drouet et al., Biotechnol. Bioeng. 43, 1075-1080, 1994; Vie et al., Enzyme Microb. Technol. 20, 597-603, 1997; Yan and Liau, Biotechnol. Lett. 20, 653-657, 1998).

Other families of enzymes have made it possible to obtain alkyl-polyglycosides.

Bousquet et al. showed the possibility of obtaining a-alkyl-polyglucosides using a-transglucosidase from Aspergillus niger (Bousquet et al., Bucke, C. (Ed.), Carbohydrate Biotechnology Protocols. Humana Press, Totowa, NJ, 291-296, 1999) or Talaromyces dupont! (Bousquet et al., Enzyme Microb. Technol. 23, 83-90, 1998). Thus, a-butyl-glucoside was obtained by transfer reaction of glucosyl units from maltodextrins onto butanol in a biphasic medium.

Dahiya and co-workers successfully produced 1-O-alkylyl-a-D-mono, di- and tri-glucopyranosides (a-alkyl-polyglycosides) from hexanol or octanol and sucrose using a strain of Microbacterium paraoxydans presenting an amylosucrase type membrane transglycosylation activity with a maximum yield of 14.8% (Dahiya et al., Biotechnol. Lett. 37, 1431-1437, 2015).

Ochs and collaborator, 2011, showed the possibility of producing pentyl- and octyl-polyxylosides (alkyl-polyxylosides) by transglycosylation reaction between pentanol or octanol and xylans from birch wood catalyzed by different xylanases including that of Thermobacillus xylanaticus (Ochs et al., Green Chem. 13, 2380-2388, 2011; Rémond et al., FR2967164, 2012).

Some Glucan Succharases (GS) of families 13 and 70 of Glycoside Hydrolases (GH13 and GH70) have been used in acceptor reactions with the aim of elongating the carbohydrate moiety of alkyl monoglucosides with a short alkyl group (1 to 8 carbon atoms) from sucrose.

GH70 GSs have also been used in direct glucosylation reactions of alcohols. Indeed, Seibel and collaborator showed the capacity of dextran saccharase from Streptococcus oralis GTFR to glucosylate alcohols ranging from (chloro-) methanol to (4-chloro)-1-butanol (Seibel et al., ChemBioChem 7, 310- 320, 2006). Furthermore, Kim and colleagues have shown, during glucosylation tests of short alcohols of 1 to 4 atoms of carbon, a preference of the dextran saccharase of L. mesenteroides B-1299CB for primary alcohols compared to secondary alcohols, the tertiary alcohols not having been able to be converted (Kim et al., Biotechnol. Lett. 31, 1433-1438 , 2009).

However, most known enzymatic processes are limited because they rarely allow the production of glycolipids having carbon chains comprising more than 8 carbon atoms.

There is therefore a need for an economical and simple process to implement on an industrial scale, which makes it possible to prepare glycolipids having new particular molecular architectures, in particular glycolipids with a bolaamphiphilic structure, which are likely to find applications in fields such as pharmacy, cosmetics, fine chemicals, biocontrol, phytosanitary, depollution and even the agri-food industry.

BRIEF DESCRIPTION OF THE FIGURES

[Fig.1] represents the screening of a-transglucosylases of the GH70 family for their ability to glucosylate 11-[(2-Hydroxy-Ethyl) Sulfanyl)]-llndecanoic acid (AHESU). Determination of the conversion rate of the lipid acceptor under the conditions tested: [AHESU] = 10 mM, [sucrose] = 292 mM, DMSO = 10% v/v; 50 mM sodium acetate buffer pH = 5.75, Enzymatic activity = 1 U.mL' ¹ , T = 30°C.

[Fig. 2] represents the screening of a-transglucosylases of the GH70 family for their ability to glucosylate 11-HydroxyUnDecanoic Acid (AHUD). Determination of the conversion rate of the lipid acceptor under the conditions tested: [AHUD] = 10 mM; [Sucrose] = 292 mM; DMSO = 10% v/v; 50 mM sodium acetate buffer pH = 5.75, Enzymatic activity = 1 U.mL' ¹ , T = 30°C.

[Fig. 3] represents the screening of a-transglucosylases of the GH70 family for their ability to glucosylate Erythro-Aleuritic Acid (EAA). Determination of the conversion rate of lipid acceptors under the conditions tested: [AEA] = 10mM; [sucrose] = 292 mM; DMSO = 10% v/v; 50 mM sodium acetate buffer pH = 5.75, Enzymatic activity = 1 U.mL' ¹ , T = 30°C. [Fig. 4] illustrates a comparison of the profiles of glucosylation products of 11 -[(2-hydroxy-ethyl) sulfanyl)]-undecanoic acid (AHESll) by the branching enzymes BRS-A, BRS-B A1, BRS-C , BRS-D A1, BRS-E and GBD-CD2 AN 123. Reactions carried out at 30°C with 800 rpm stirring in the presence of 438.5 mM sucrose, 10 mM AHESU, 10% v/v DMSO and 50 mM sodium acetate buffer at pH=5.75. Numbers 1 to 6 represent the major glucosylation products. The enzymes are grouped according to their specificity of saccharide binding on dextrans ie a-1,2 for BRS-A, BRS-D A1, GBD-CD2 AN123 or a-1,3 for BRS-B A1, BRS-C , BRS-E A1 ).

[Fig. 5] illustrates a comparison of the profiles of 11-hydroxy-undecanoic acid (AHUD) glucosylation products synthesized by the branching enzymes BRS-A, BRS-B A1, BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN 123. Reactions carried out at 30°C with 800 rpm stirring in the presence of 438.5 mM sucrose, 10 mM AHUD, 10% v/v DMSO and sodium acetate buffer 50 mM at pH=5.75. The enzymes are grouped according to their specificity of saccharide bond synthesis on dextrans i.e. a-1,2 for BRS-A, BRS-D A1, GBD-CD2 AN123 or a-1,3 for BRS-B A1, BRS -C, BRS-E A1.

[Fig.6] represents a comparison of the profiles of erythro-aleuritic acid (EAA) glucosylation products synthesized by the enzymes BRS-A, BRS-B A1, BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN123. Reactions carried out at 30°C with 800 rpm stirring in the presence of 438.5 mM sucrose, 10 mM AEA, 10% v/v DMSO and 50 mM sodium acetate buffer at pH=5.75. Numbers 1 to 16 represent peaks of major glucosylation products. The enzymes are grouped according to their specificity of saccharide bond synthesis on dextrans i.e. a-1,2 for BRS-A, BRS-D A1, GBD-CD2 AN123 or a-1,3 for BRS-B A1, BRS -C, BRS-E A1.

[Fig. 7] represents the conversion rate of 11-[(2-hydroxy-ethyl) sulfanyl)]- undecanoic acid (AHESU) obtained during its glucosylation by the branching enzymes BRS-A, BRS-B A1, BRS- C, BRS-D A1, BRS-E A1 and GBD-CD2 AN123 as a function of initial sucrose concentration. Reactions carried out at 30°C with 800 rpm stirring in the presence of increasing concentrations of sucrose; 10 mM 11-[(2-hydroxy-ethyl)sulfanyl)]-undecanoic acid; 10% v/v DMSO and 50 mM sodium acetate buffer at pH=5.75. [Fig. 8] represents the conversion rate of AHUD obtained during its glucosylation by the branching enzymes BRS-A, BRS-B A1, BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN 123 into function of the initial sucrose concentration. Reactions carried out at 30°C with 800 rpm stirring in the presence of increasing concentrations of sucrose; 10 mM AHUD; 10% v/v DMSO and 50 mM sodium acetate buffer at pH=5.75.

[Fig. 9] represents the conversion rate of erythro-aleuritic acid (AEA) obtained during its glucosylation by the branching enzymes BRS-A, BRS-B A1, BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN 123 as a function of the initial sucrose concentration. Reactions carried out at 30°C with 800 rpm stirring in the presence of increasing concentrations of sucrose; 10 mM AEA; 10% v/v DMSO, 1 II. ^mL'1 of enzymatic activity in reaction and a 50 mM sodium acetate buffer at pH=5.75.

[Fig. 10] represents a chromatogram of the glucosylation products of 11-[(2-hydroxy-ethyl) sulfanyl)]-undecanoic acid (AHESll) obtained with the BRS-A enzyme after elimination of free sugars by chromatography on Purasorb PAD910 resin .

[Fig. 11] represents a chromatogram of the glucosylation products of 11-hydroxyundecanoic acid (AHIID) after elimination of free sugars by flash chromatography and mass spectrometry analysis of the purified products corresponding to peaks 1 and 2 (glucosylation products no. 1 and No. 2).

[Fig. 12] represents a chromatogram of the glucosylation products of erythro-aleuritic acid (EAA) after pre-purification (elimination of free sugars) by flash chromatography and mass spectrometry analysis of the purified products.

[Fig.13A] [Fig. 13B] [Fig.13C] represent the glucosylation profile of 12-hydroxydodecanoic acid (AHDD) ([Fig.13A]), 15-hydroxypentadecanoic acid (AHPD) ([Fig. 13B]) and l 16-hydroxy-hexadecanoic acid (AHHD) ([Fig. 13C]) by the branching enzymes BRS-A, BRS-B A1, BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN123.

[Fig. 14A] [Fig. 14B] represent the glucosylation profile of 9, 10 dihydroxyoctanoic acid (ADHO) (Figure 14A) and 9, 10, 12 trihydroxyoctadecanoic acid (ATHO) (Figure 14B) by the branching enzymes BRS-A, BRS -B A1, BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN 123. Reactions carried out at 37°C under 800 rpm stirring in the presence of 877 mM sucrose, 5 mM fatty acid, an enzymatic activity of 1 ^U.mL'1 , 10% v/v DMSO and a 50 mM sodium acetate buffer at pH= 5.75. The enzymes are grouped according to their specificity of saccharide bond synthesis on dextrans ie a-1,2 for BRS-A, BRS-D A1, GBD-CD2 AN123 or a-1,3 for BRS-B A1, BRS -C, BRS-E A1.

[Fig. 15] illustrates the characterization at OJ of emulsions consisting of an aqueous solution of 2% Glc-AHESU and 20% by weight of dodecane, at the different pHs studied. The D(4.3) represents the volume average diameter and P the polydispersity.

[Fig. 16] illustrates the stability monitoring over 14 days of emulsions whose aqueous phase includes 2% by weight of Glc-AHESU (O/W 20/80). The size of the drops is determined by particle size and confirmed by optical microscopy.

[Fig. 17]: Study of the destabilization of the emulsion (2% Glc-AHESU in water/dodecane 80/20). On the left, the emulsion is manufactured and maintained at pH=6. On the right, the emulsion is manufactured at pH=6 then adjusted to pH=2 using a 0.1 M HCl solution.

[Fig.18] illustrates the characterization on D0 of the emulsions consisting of an aqueous solution of 2% Glc-AHUD and 20% by weight of dodecane, at the different pHs studied. The D(4.3) represents the volume average diameter and P the polydispersity.

[Fig. 19]: Monitoring the stability over several weeks of emulsions containing 2% surfactant (O/W 20/80). The size of the drops is determined by particle size and confirmed by optical microscopy.

[Fig. 20] illustrates the destabilization of the emulsion (2% GAH in water/Dodecane 80/20). On the left, the emulsion is manufactured and maintained at pH=6. On the right, the emulsion is manufactured at pH=6 then adjusted to pH=2 using a 0.1 M HCI solution. GAH = glucosylated form of AHED

[Fig. 21] represents the influence of the hydrophobicity of the support, the nature of the oil, evaporation, the size and concentration of the drops on the intensity of the phenomena observed during the deposition of a drop of emulsion on a solid support. [Fig. 22] shows the macroscopic appearance of the primary emulsion (a), the macroscopic appearance of the dry emulsion (powder) (b), the microscopic observation of the emulsion before drying (c), the microscopic observation of the emulsion after drying and redispersion in water (d); measuring the droplet size distribution before drying and after drying and redispersion in water (e).

[Fig. 23] represents the monitoring of cultures of E. coli K12 (OD 600 nm) in the presence of concentrations of 0% w/v (O), 0.5% w/v (□), 1% w/v (A), 2% w/v (X) and 2.5% w/v (*) of glucosylation products (A) of AHUD and AEA (B) in aqueous phase.

BRIEF DESCRIPTION ACCORDING TO THE INVENTION

An aim of the present invention is to overcome the drawbacks of the prior art and to provide a process for preparing a glycolipid of formula (I) by enzymatic catalysis.

Surprisingly, the inventors discovered that a-transglucosylases are capable of glucosylating hydroxylated fatty acids.

An advantage of the process according to the invention is that it uses renewable and inexpensive substrates, namely sucrose or one of its analogues and biosourced hydroxylated fatty acids.

The process also has the advantage that it makes it possible to obtain glycolipids having a bolaamphiphilic type structure (i.e. made up of two polar parts separated by a hydrophobic part) which suggests interesting surfactant and biological properties. The glycolipids according to the invention are, however, distinguished from sophorolipids in that their saccharide unit can comprise a single glucosyl unit or several glucosyl units connected by a-bonds whose nature can be modulated (a-1, 2 and/or a- 1,3 and/or a-1,4 and/or a-1,6) depending on the a-transglucosylase used.

Importantly, the process according to the invention makes it possible to prepare bolaamphiphilic glycolipids whose hydrophobic part comprises a carbon chain of large size, typically greater than 8 carbon atoms, which can be interrupted by at least one sulfanyl group, and substituted for example by at least one hydroxyl group. The process according to the invention therefore makes it possible to obtain a great diversity of glycolipids in terms of the size of their hydrophobic part and the structure of their glucosidic part.

This diversity is very advantageous for producing new glycolipids of interest useful in fields such as pharmacy, cosmetics, fine chemicals, biocontrol, phytosanitary, depollution or even the agri-food industry.

Another aim according to the invention is to provide glycolipids, in particular bolaamphiphilic glycolipids of a wide diversity of molecular architecture, and which can be useful as a surfactant or an antimicrobial agent, in particular an antibacterial agent.

These goals are achieved by the invention which will be described below.

Thus, an object according to the invention relates to a process for preparing at least one glycolipid corresponding to formula (I):

[Glc] _n -xOy-R (I) in which

[Glc] _n represents a linear or branched saccharide unit comprising n glucosyl units, with n between 1 and 7, with the condition that when the saccharide unit comprises several glucosyl units these are linked together by type a saccharide bonds ;

R represents a fatty acid radical comprising between 4 and 24 carbon atoms, the carbon chain of which is linear or branched, saturated or unsaturated, optionally interrupted by one or more sulfur atoms, optionally still being able to comprise one or more chosen substituent(s) (s) from the hydroxyl group, the carbonyl group, the methoxyl group, the amine group, the nitrosyl group, and the thiol group,

-xOy- symbolizes the attachment of the fatty acid radical to the saccharide unit by an ether bond connecting a Cx carbon atom of a glucosyl residue of the saccharide unit, previously carrying a hydroxyl group, to a Gy carbon atom of the fatty acid radical, previously carrying a hydroxyl group, with Cx the position of the atom of the glucosyl residue on which the bond takes place, Cx representing the C1 carbon atom of the glucosyl residue, Cy being a carbon atom positioned along the carbon chain of the fatty acid radical or at the omega end thereof; said process comprising a step of glucosylation of a hydroxylated fatty acid of formula (II):

R-yOH (II) in which

R is a fatty acid radical as defined in formula (I),

-yOH represents a hydroxyl group attached to a Cy carbon atom as defined above; said step comprising bringing the hydroxylated fatty acid of formula (II) into contact with at least one a-transglucosylase of the GH70 family in the presence of sucrose or a sucrose analogue.

Another object according to the invention relates to a glycolipid corresponding to formula (I): [Glc] _n -xOy-R (I) in which

-xOy- symbolizes the attachment of the fatty acid radical to the saccharide unit by an ether bond connecting a Cx carbon atom of a glucosyl residue of the saccharide unit, previously carrying a hydroxyl group, to a Cy carbon atom of the fatty acid radical, previously carrying a hydroxyl group, with Cx the position of the atom of the glucosyl residue on which the bond takes place, Cx representing the C1 carbon atom of the glucosyl residue, Cy being a carbon atom positioned along the carbon chain of the fatty acid radical or at the omega end thereof.

Another object according to the invention concerns the use of a glycolipid according to the invention, as a surfactant. Another object according to the invention concerns the use of a glycolipid according to the invention as defined above, as an antimicrobial agent.

Another object according to the invention relates to an oil-in-water emulsion comprising an oily phase dispersed in an aqueous phase and at least one glycolipid of formula (I) according to the invention, characterized in that the emulsion is kinetically stable when the pH of the aqueous phase is greater than a threshold hydrogen potential pH _s advantageously between 2 and 5.

Another subject concerns the use of an emulsion according to the invention for cleaning surfaces.

DETAILED DESCRIPTION ACCORDING TO THE INVENTION

Process for preparing at least one glycolipid according to the invention

In the context of the present invention, the expressions “glycolipid of formula (I)”, “glycolipid”, “glycolipid according to the invention” can be used interchangeably. In this document, the terms "glycolipid" and "glucolipid" may be used interchangeably.

In the context of the present invention, the expressions “hydroxylated fatty acid of formula (II)”, “hydroxylated fatty acid” and “hydroxylated fatty acid according to the invention” can be used interchangeably.

Osidic pattern

According to the invention, the expression “saccharide unit” designates a linear or branched polymer consisting of glucosyl units linked together by saccharide bonds.

According to the invention, the terms “osidic bond” or “glucosidic bond” can be used interchangeably and designate a covalent bond which links a glucosyl to another adjacent glucosyl within the saccharide unit. The [Glc] _n saccharide motif corresponds to the n glucosyl units grafted during the glucosylation step at the Cy carbon of the hydroxylated fatty acid according to the invention by the a-transglucosylase of the GH70 family. n represents the number of glucosyl units in the [Glc] _n saccharide unit of the glycolipid according to the invention, n is advantageously between 1 and 7, preferably between 1 and 4. Preferably, n is equal to 1 or 2.

In a particular embodiment, the glycolipid according to the invention is a glycolipid whose saccharide unit comprises several glucosyl units. Advantageously, the glycolipid according to the invention is such that n is between 2 and 7, preferably between 2 and 4.

Bonds within the saccharide motif

According to the invention, the expression “a bond” designates a covalent bond which binds the C1 carbon atom of a glucosyl unit in its a configuration; the expression “0 bond” designates a covalent bond that binds the C1 carbon atom of a glucosyl unit in its 0 configuration.

When the saccharide unit comprises several glucosyl units, these are linked together by an α saccharide bond. Advantageously, the choice of the a-transglucosylase type of the GH70 family makes it possible to modulate the type of bond within the saccharide motif.

The saccharide bond(s) within the saccharide unit are advantageously chosen from an a-1,2 saccharide bond, an a-1,3 bond, an a-1,4 bond or a bond a-1,6, or a mixture thereof; preferably also chosen from an a-1,2 bond, an a-1,3 bond, an a-1,4 bond, an a-1,6 bond or a mixture of these.

According to the invention, the term "a-1,3 bond" designates the covalent ether bond which links the C1 carbon atom of a glucosyl unit formerly carrying the hemi-acetal function in its a configuration and the hydroxyl carried by carbon 03 of another adjacent glucosyl unit. According to the invention, the term “a-1,2 bond” designates the covalent ether bond which binds the carbon atom 01 of a glucosyl unit formerly carrying the hemi-function. acetal in its a configuration and the hydroxyl carried by the C2 carbon of another adjacent glucosyl unit. According to the invention, the term "a-1,4 bond" designates the covalent ether bond which links the C1 carbon atom of a glucosyl unit formerly carrying the hemi-acetal function in its a configuration and the hydroxyl carried by the C4 carbon of another adjacent glucosyl unit. According to the invention, the term "a-1,6 bond" designates the covalent ether bond which links the C1 carbon atom of a glucosyl unit formerly carrying the hemi-acetal function in its a configuration and the hydroxyl carried by the C6 carbon of another adjacent glucosyl unit.

The α-glucosyl units are preferably α-D-glucosyl units.

The analysis of saccharide bonds can be carried out by methods known to those skilled in the art.

For example, the osidic bonds within the osidic motif can be analyzed by nuclear magnetic resonance (NMR) or mass spectrometry (MS).

Bond between the saccharide unit and the fatty acid radical

-xOy- symbolizes the attachment of the fatty acid radical to the saccharide unit by an ether bond connecting a Cx carbon atom of a glucosyl residue of the saccharide unit, previously carrying a hemiacetal group, to a Cy carbon atom of the fatty acid radical, previously carrying a hydroxyl group, with Cx the position of the atom of the glucosyl residue on which the bond takes place, Cx representing the C1 carbon atom of the glucosyl residue, Cy being a carbon atom positioned along the carbon chain of the fatty acid radical or at the omega end thereof.

For the purposes of the present invention, the position of the carbon atoms of a glucosyl unit is numbered so that the C1 carbon atom of a glucosyl unit is the carbon atom which carries the aldehyde -CHO function under the open form of this glucosyl unit.

For the purposes of the present invention, the position of the carbon atoms of the fatty acid radical is numbered so that C'1 represents the carbon atom of the carboxylic acid function of the fatty acid radical. For the purposes of the present invention, the omega end of the fatty acid radical is the end terminated by a carbon called omega carbon whose position is opposite to the carbon carrying the carboxylic acid group of the fatty acid radical.

Within the glycolipid according to the invention, the glucosyl unit adjacent to the fatty acid radical is advantageously linked to the fatty acid radical by an α bond.

In other words, in the glycolipid of formula (I), the glucosyl unit adjacent to the fatty acid radical is preferably in the a configuration.

The analysis of the bond between the saccharide motif and the fatty acid radical can be carried out using any method known to those skilled in the art, for example by nuclear magnetic resonance (NMR).

Radical fatty acid

Advantageously, the fatty acid radical comprises at least 6, 7, 8, 9, 10, or 11 carbon atoms. Advantageously, the fatty acid radical comprises at most 24 carbon atoms. Advantageously, the fatty acid radical comprises between 6 and 24 carbon atoms, between 7 and 24 carbon atoms, between 8 and 24 carbon atoms, between 9 and 24 carbon atoms, between 10 and 24 carbon atoms, preferably between 11 and 24 carbon atoms, preferably between 11 and 20 carbon atoms.

According to a particular embodiment, the radical R can be represented by the following general formula:

in which o the wavy line*^ represents the point of attachment to -xOy- o C _y is as defined above and o R ^a represents H or a carbon chain comprising between 1 and 22 carbon atoms, said chain being linear or branched, saturated or unsaturated, optionally interrupted by one or more sulfur atoms, optionally still being able to comprise one or more substituent(s) chosen from the hydroxyl group, the carbonyl group, the methoxyl group, the amine group, the nitrosyl group, and the thiol group, o R ^b represents a carbon chain comprising between 1 and 22 carbon atoms, said chain being linear or branched, saturated or unsaturated, optionally interrupted by one or more sulfur atoms, optionally still being able to comprise one or more substituent(s) chosen from the hydroxyl group, the carbonyl group, the methoxyl group, the amine group, the nitrosyl group, and the thiol group, o it being understood that the sum of the carbon atoms included in the two radicals R ^a and R ^b is between 2 and 22, and preferably between 4 and 22.

Preferably, R ^a represents H (case where Cy is the carbon at the omega end of the fatty acid radical) or a linear carbon chain, optionally interrupted by one or more sulfur atoms, optionally still being able to comprise one or more substituent(s). ) chosen from the hydroxyl group, the carbonyl group, the methoxyl group, the amine group, the nitrosyl group, and the thiol group (case where Cy is a carbon positioned along the carbon chain of the fatty acid radical) and R ^b represents a linear carbon chain, optionally interrupted by one or more sulfur atoms, optionally still being able to comprise one or more substituent(s) chosen from the hydroxyl group, the carbonyl group, the methoxyl group, the amine group, the nitrosyl group, and the thiol group.

According to a particular embodiment, the carbon chain of the fatty acid radical is linear, that is to say that Cy is the carbon at the omega end of the fatty acid radical. In this embodiment, R ^a represents H and R ^b represents a linear carbon chain comprising between 2 and 22 carbon atoms and preferably between 4 and 22 carbon atoms, optionally interrupted by one or more sulfur atoms, optionally still being able to comprise one or more substituent(s) chosen from the hydroxyl group, the carbonyl group, the methoxyl group, the amine group, the nitrosyl group, and the thiol group.

According to a preferred embodiment, when the fatty acid radical comprises an amine substituent (NH or NH2 group), this amine group is not located in position a relative to the carboxyl function. Preferably, the possible substituent(s) of the carbon chain is(are) chosen from the hydroxyl group, the carbonyl group, the methoxyl group or the thiol group, more preferably from the hydroxyl or thiol group.

Typically, the carbon chain (or radical) R optionally comprises at most three hydroxyl groups, preferably at most three substituents chosen from the hydroxyl group, the carbonyl group, the methoxyl group, the amine group, the nitrosyl group, and the thiol group, preferably chosen from the hydroxyl group, the carbonyl group, the methoxyl group, and the thiol group, preferably at most three substituents. More specifically: when the carbon chain comprises between 4 and 11 carbon atoms (preferably between 6 and 11 carbon atoms), said carbon chain comprises at most one hydroxyl group, preferably at most one substituent chosen from the hydroxyl group, the carbonyl group, methoxyl group, and thiol group, preferably at most one substituent; when the carbon chain comprises between 12 and 16 carbon atoms, said carbon chain comprises at most two hydroxyl groups, preferably at most two substituents chosen from the hydroxyl group, the carbonyl group, the methoxyl group, and the thiol group, preferably at most two substituents; when the carbon chain comprises between 17 and 24 carbon atoms said carbon chain comprises at most three hydroxyl groups, preferably at most three substituents chosen from the hydroxyl group, the carbonyl group, the methoxyl group, and the thiol group, preferably at plus three substituents.

Particularly advantageously: i) the radical R can be represented by the following general formula:

in which the wavy line

, Cy, R ^a and R ^b are as defined above (any of the embodiments described above); and ii) R ^a and R ^b together optionally comprise at most three hydroxyl groups (in other words, at most three hydroxyl groups are distributed over R _a and Rb), preferably at most three substituents chosen from the hydroxyl group , the carbonyl group, the methoxyl group, the amine group, the nitrosyl group, and the group thiol, preferably chosen from the hydroxyl group, the carbonyl group, the methoxyl group, and the thiol group, preferably at most three substituents; and iii) when R ^a or R ^b comprises an amine substituent (NH or NH2 group), this amine group is not located in position a relative to the carboxyl function.

In this particularly preferred embodiment, characteristic ii) is preferably such that: o when R ^a and R ^b together comprise between 2 and 9 carbon atoms (preferably between 4 and 9 carbon atoms), R ^a and R ^b together comprise at most one hydroxyl group, preferably at most one substituent chosen from the hydroxyl group, the carbonyl group, the methoxyl group, and the thiol group, preferably at most one substituent; o when R ^a and R ^b together comprise between 10 and 14 carbon atoms, R ^a and R ^b between them comprise at most two hydroxyl groups, preferably at most two substituents chosen from the hydroxyl group, the carbonyl group, the methoxyl group, and the thiol group, preferably at most two substituents; o when R ^a and R ^b between them comprise between 15 and 22 carbon atoms R ^a and R ^b between them comprise at most three hydroxyl groups, preferably at most three substituents chosen from the hydroxyl group, the carbonyl group, the methoxyl group, and the thiol group, preferably at most three substituents.

Glycolipid blend

In one embodiment, the process according to the invention allows the preparation of a mixture of glycolipids according to the invention. Advantageously, the mixture contains glycolipids according to the invention which differ only in the number of glucosyl units of their saccharide unit and possibly in the nature of the saccharide bond.

Preferably, the mixture essentially consists of glycolipids in which n is between 1 and 7, preferably n is between 1 and 4, preferably in which n is equal to 1, 2 or 3, more preferably in which n is equal to 1 or 2. Preferably, the mixture of glycolipids according to the invention comprises from 70% to 100%, preferably from 90% to 100% by weight of glycolipids according to the invention in which n is between 1 and 7, preferably n is between 1 and 4, preferably in which n is equal to 1, 2 or 3, more preferably in which n is equal to 1 or 2, in % by weight relative to the total weight of glycolipids of the mixture.

Hydroxy fatty acids

The process according to the invention is carried out with a hydroxylated fatty acid of formula (II) R-yOH (H) in which

R is a fatty acid radical as defined in formula (I),

-yOH- represents a hydroxyl group attached to a Cy carbon atom as defined above.

The process can in particular be implemented with three categories of hydroxylated fatty acids.

1st variant: sulfanylated hydroxy fatty acid

According to a first variant, the process according to the invention is carried out with a sulfanylated hydroxy fatty acid.

In this embodiment, the hydroxylated fatty acid advantageously corresponds to the formula (HA):

HO-(CH ₂ )bS-(CH ₂ ) _a -COOH (HA) in which a is between 9 and 14, b is greater than or equal to 2, and with the sum of a and b less than 23.

The hydroxylated fatty acid of formula (HB) is for example 11-[(2-hydroxyethyl)sulfanyl]-undecanoic acid OH-(CH ₂ ) ₂ -S-(CH ₂ )IO-COOH.

2nd variant: hydroxylated fatty acid in the omega position

According to a second variant, the process according to the invention is carried out with a fatty acid hydroxylated in the omega position. In one embodiment, the hydroxylated fatty acid corresponds to the formula (I IB):

OH-(CH ₂ ) _a -COOH (IIB) in which a is between 3 and 23, preferably between 10 and 15.

Advantageously, a is between 11 and 15.

The hydroxylated fatty acid of formula (IIB) is for example 11-hydroxy-undecanoic acid OH-(CH ₂ )IO-COOH.

3rd variant: Poly-hydroxy fatty acid

According to a 3rd variant, the process according to the invention is carried out with a poly-hydroxy fatty acid, preferably chosen from a di-hydroxy fatty acid and a tri-hydroxy fatty acid.

Preferably, the di-hydroxy fatty acid is: a di-hydroxy fatty acid whose carbon chain is substituted by two hydroxyl groups positioned along the carbon chain, for example 9, 10-dihydroxy-octadecanoic acid, or a di-hydroxy fatty acid whose carbon chain is substituted by a hydroxyl group positioned along the chain and by a hydroxyl group positioned at the omega end thereof.

Advantageously, the tri-hydroxy fatty acid is: a tri-hydroxy fatty acid whose carbon chain is substituted by two hydroxyl groups positioned along the carbon chain and by a hydroxyl group in the omega position thereof, for example l erythro-aleuritic acid, or a tri-hydroxy fatty acid whose carbon chain is substituted by three groups positioned along the carbon chain, for example 9, 10, 12-trihydroxy-octadecanoic acid or 9, 10,12-trihydroxystearic. g-transglucosylase

The inventors have, for the first time, shown that a-transglucosylases of the GH70 family are capable of catalyzing the glucosylation of hydroxylated fatty acids from sucrose. The inventors have further shown that, unexpectedly, this glucosylation is particularly effective when the a-transglucosylases of the GH70 family used are branching saccharases of the GH70 family.

According to the invention, the term "a-transglucosylase" designates an enzyme capable of polymerizing glucosyl units along α-linkages, by catalyzing the transfer of a glucosyl unit from a glucosyl donor sugar to a hydroxylated acceptor compound.

According to the invention, the expression "of the GH70 family", relating to a-transglucosylase, means that the a-transglucosylase according to the invention belongs to family 70 of glycosidehydrolases according to the Q Z classification (www.cazy. org). CAZy, from the English "Carbohydrate-Active enZYmes" (abbreviated "CKZ\/"), is a bioinformatics database for the classification of enzymes active on sugars i.e. capable of catalyzing their dissociation or their synthesis according to sequence homologies or of structure, in particular of their catalytic and carbohydrate-binding modules. In the CAZy classification, the group of glycoside hydrolases (GH) includes 173 families composed of enzymes active on sugars capable of catalyzing hydrolysis reactions of glycosidic bonds or transglucosylation.

The a-transglucosylases of the GH70 family are typically produced naturally by lactic acid bacteria of the genera Streptococcus, Leuconostoc (abbreviated “L.”), Weisella, Oenococcus or Lactobacillus (abbreviated “Lb.”).

The a-transglucosylases of the GH70 family according to the invention are active on sucrose, which means that the a-transglucosylases according to the invention specifically use sucrose or one of its analogues as a glucosyl donor.

Advantageously, the sucrose analogs are compounds of formula (I): X-a-D-glucopyranosyl (I) in which -p-D-fructofuranosyl-(2->1) and a-L-sorbofuranoside.

According to the present invention, the LG group represents a chemical group which can be easily displaced by a nucleophile during a nucleophilic substitution reaction, the nucleophile being more particularly a group originating from the a-transglucosylase of the GH70 family. Such a leaving group may more particularly be a halogen atom such as a chlorine, bromine or fluorine atom, a tosylate group (-OS(O)2-(p-Me-CeH4)), or a group optionally substituted aromatic, in particular an -O-C6H5-NO2 group

(preferably p-nitrophenyl or o-nitrophenyl), a 4-Methylumbelliferyl group

resorufin group (phenoxazine 3-one group substituted at position 7) or a group

For the purposes of the present invention, an “aromatic” group means an aryl or heteroaryl group optionally substituted, preferably by 1 to 2 substituents, in particular chosen from a nitro group (-NO2), a halogen, an alkyl group in C1-C4 or an optionally substituted aryl.

By “aryl” is meant, for the purposes of the present invention, an aromatic hydrocarbon group, preferably comprising from 6 to 14 carbon atoms, and comprising one or more attached rings, for example a phenyl or naphthyl group. Advantageously, it is phenyl.

By “heteroaryl” or “heteroaromatic” is meant, within the meaning of the present invention, an aromatic group comprising 5 to 14 cyclic atoms including one or more heteroatoms, advantageously 1 to 4 and even more advantageously 1 or 2, such as for example nitrogen or oxygen atoms, the other cyclic atoms being carbon atoms. Examples of heteroaryl groups are indyl, umbelliferyl or resorufin.

By “halogen” is meant, within the meaning of the present invention, the atoms of fluorine, chlorine, bromine and iodine. Preferably, it is a chlorine or fluorine atom. By “(C1-C4) alkyl” group is meant, for the purposes of the present invention, a saturated monovalent hydrocarbon chain, linear or branched, comprising 1 to 4 carbon atoms, and preferably methyl.

An aryl is preferentially substituted by one or two substituents preferably chosen from a nitro group (-NO2), a halogen, a C1-C4 alkyl group and/or a -C(O)-heteroaryl group, such as a group 2-acyl-N-methylpyrrole.

Thus, the sucrose analogs can in particular be chosen from the group comprising a-D-glucopyranosyl fluoride (in English "a-D-glucopyranosyl fluoride"), O-p-D-galactopyranosyl-(1 ->-4)-p-D-fructofuranosyl- (2->1)-a-D-glucopyranoside (in English “Lactulosucrose”), p-nitrophenyl a-D-glucopyranoside, a-D-glucopyranosyl a-L-sorbofuranoside, 4-Methylumbelliferyl a-D-glucosaminide (or 4-methylumbelliferyl 2-Amino- 2-deoxy-a-D-glucopyranoside, CAS no. [137687-00-4]), resorufin a-D-glucopyranoside (CAS no.: [136565-96-3]), Aldol® 484 alpha-D-glucopyranoside ( CAS number: [2484872-56-0]) and their mixtures. The sucrose analogs may in particular be chosen from the group comprising a-D-glucopyranosyl fluoride (in English “a-D-glucopyranosyl fluoride”), O-p-D-galactopyranosyl-(1^4)-p-D-fructofuranosyl-(2— >1)-a-D-glucopyranoside (in English “Lactulosucrose”), p-nitrophenyl a-D-glucopyranoside, a-D-glucopyranosyl a-L-sorbofuranoside and their mixtures.

Sucrose is preferred because this substrate is biosourced and inexpensive.

Advantageously, the a-transglucosylase of the GH70 family is a branching saccharase of the GH70 family, a glucan-sucrase of the GH70 family, or a mixture of these.

In this document, the terms “branching sucrase of the GH70 family” (in English “branching sucrase”, sometimes abbreviated BRS) or “a-transglucosylase of the GH70 family with branching activity” or “branching enzyme of the family GH70" are used interchangeably and refer to an a-transglucosylase of the GH70 family capable of catalyzing the addition of glucosyl units in the main chain of a pre-existing dextran type glucan forming branches (or in other words branches ). The nature of the branching connection (connections a-1,2; a-1,3; a-1,4 or a- 1, 6) and the length of the chains of glucosyl units constituting the branches vary depending on the specificity of the branching saccharase considered.

Preferably, the branching saccharase of the GH70 family has as its amino acid sequence a sequence chosen from the group comprising GBD-CD2 AN123 (SEQ ID NO: 1), BRS-A (SEQ ID NO: 2), BRS- B A1 (SEQ ID NO: 3), BRS-C (SEQ ID NO: 4), BRS-D A1 (SEQ ID NO: 5), BRS-E A1 (SEQ ID NO: 6), or a sequence of amino acids having at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with at least minus one of the sequences SEQ ID NO: 1 to SEQ ID NO: 6.

The inventors further showed that mutants of GBD-CD2AN123 (SEQ ID NO: 1) were particularly effective for the glucosylation of hydroxylated fatty acids. Preferably, the branching saccharase of the GH70 family is a mutant of GBD-CD2AN123 (SEQ ID NO: 1) having an amino acid sequence chosen from GBD-CD2 AN123 W2135L F2136L (SEQ ID NO: 7), GBD -CD2 AN123 W2135L (SEQ ID NO: 8), GBD-CD2 AN123 W2135I F2136Y (SEQ ID NO: 9), GBD-CD2 AN123 W2135I F2136C (SEQ ID NO: 10), GBD-CD2 AN123 W2135V (SEQ ID NO: 11), or an amino acid sequence having at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % sequence identity with at least one of the sequences SEQ ID NO: 7 to SEQ ID NO: 11.

In the present document, the expression "glucan-sucrase of the GH70 family" sometimes abbreviated "GS" refers to an a-transglucosylase of the GH70 family capable of catalyzing the synthesis of a-glucans, ie of polysaccharides composed exclusively of glucosyl units linked together by a-linkages. Among the glucan-sucrases, we can cite dextran-sucrases (sometimes abbreviated DSR), which synthesize dextrans, ie glucans whose main chain residues are mainly linked at a-1,6; reuteran-sucrases, which synthesize reuterans, ie glucans whose main chain residues are linked at a-1,4 and a1,6; mutane-sucroses, which synthesize mutanes, ie glucans whose main chain residues are mainly linked in a-1,3; alternan-sucrases, ie glucans whose main chain residues are linked alternately at a-1,3 and a-1,6. Preferably, the glucan-sucrase of the GH70 family has as its amino acid sequence a sequence chosen from the group comprising DSR-OK (SEQ ID NO: 12), DSR-M DP (SEQ ID NO: 13), DRS- S vardelA4N (SEQ ID NO: 14), ASR Cdelbis-Athio (SEQ ID NO: 15), GTF-SI (SEQ ID NO: 16), GTF-J (SEQ ID NO: 17), DSR-M A1 (SEQ ID NO: 18), or an amino acid sequence having at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% sequence identity with at least one of the sequences SEQ ID NO: 12 to SEQ ID NO: 18.

As used herein, the term "sequence identity" or "identity" refers to the number (%) of matches (identical amino acid residues) to positions originating from an alignment of two polypeptide sequences. Sequence identity is determined by comparing sequences when aligned in a manner that maximizes overlap and identity while minimizing sequence interruptions. In particular, sequence identity can be determined using any of several global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using global alignment algorithms (eg Néedleman & Wunsch, J. Mol. Biol 48:443, 1970) which align sequences optimally over the entire length, while sequences of similar lengths of significantly different lengths are preferably aligned using a local alignment algorithm, for example the Smith and Waterman algorithm (Smith and Waterman, Adv. Appl. Math. 2:482, 1981) or the AltschuI algorithm (Altschul et al (1997) Nucleic Acids Res. 25:3389-3402; Altschul et al (2005) FEBS J. 272:5101-5109). The alignment for the purpose of determining the percentage of amino acid sequence identity can be carried out by any method known to those skilled in the art, for example by using software available on Internet sites such as http: // blast. ncbi.nlm. nih.gov / or http://www.ebi.ac.uk/Tools/emboss/. Those skilled in the art can easily determine the appropriate parameters for measuring alignment. For the purposes of the present invention, amino acid sequence identity percentage values refer to values generated using the "Protein BLAST" (or Blastp) program in which the parameters are the default parameters (expected threshold (in English “Expect threshold”): 10, Word size: 6, Matrix = BLOSUM62, Gap Costs: Existence = 11, Extension = 1, Conditional compositional score matrix adjustment. Implementation of the process

In one embodiment, step i) is carried out in solution at a controlled pH, in particular by the use of a buffer solution.

Advantageously, step i) is carried out at a pH value of between 5 and 8.

Step i) is advantageously carried out with an initial concentration of sucrose or a sucrose analogue of between 100 and 650 ^g.L'1 .

Step i) is preferably carried out with a molar ratio of sucrose or sucrose analogue: hydroxylated fatty acid of formula (II) of between 1 and 100, preferably between 10 and 100.

Preferably, step i) is carried out at a temperature between 10°C and 80°C.

Advantageously, step i) is carried out with an a-transglucosylase of the GH70 family in solution, suspension or immobilized.

When a glycolipid mixture is obtained at the end of step (i), the process may also comprise a step ii) of purification of one or more glycolipid(s) of formula (I).

Glycolipid

A second object according to the invention relates to a glycolipid of formula (I) as defined in the first object according to the invention.

The glycolipids according to the invention have the advantage of being biodegradable.

Advantageously, the glycolipid of the second object according to the invention is a glycolipid in which n is greater than or equal to 2, preferably between 2 and 7, more preferably between 2 and 4. Advantageously, the glycolipid of the second object according to the invention is a glycolipid in which the fatty acid radical comprises at least 6, 7, 8, 9, 10, or 11 carbon atoms. Advantageously, the fatty acid radical comprises between 10 and 24 carbon atoms, preferably between 11 and 24 carbon atoms, preferably between 11 and 20 carbon atoms.

1st variant: glycolipid comprising a sulfanylated carbon chain

According to a first variant, the glycolipid comprises a sulfanylated carbon chain. In this embodiment, the glycolipid advantageously corresponds to formula (IA): [Glc] _n -xOy-(CH ₂ )bS-(CH ₂ )a-COOH in which a is between 9 and 14 b is greater or equal to 2 the sum of a and b is less than 23.

Advantageously, the glycolipid of formula (IA) corresponds to the formula [Glc] _n -xOy-(CH ₂ ) ₂ -S-(CH ₂ )io-COOH

2nd variant: Glycolipid comprising an alkyl type carbon chain

In a second variant according to the invention, the glycolipid of formula (I) comprises a carbon chain of the alkyl type, advantageously of the linear alkyl type.

In this embodiment, the glycolipid of formula (I) advantageously corresponds to formula (IB):

[Glc]n-xOy-(CH ₂ ) _a -COOH (IB) in which a is between 3 and 23, preferably between 10 and 15.

Advantageously, the glycolipid of formula (IB) corresponds to the formula [Glc] _n -xOy-(CH ₂ )io-COOH.

3rd variant: Glycolipid comprising a hydroxylated carbon chain

In a third variant according to the invention, the fatty acid radical is substituted by one or two hydroxyl groups. In this embodiment, the glycolipid of formula (I) is preferably a glycolipid in which: the carbon atom Cy is a carbon atom positioned at the omega end, and the carbon chain of the fatty acid radical is substituted by one or two hydroxyl groups positioned along the carbon chain, or the carbon atom Cy is a carbon atom positioned along the carbon chain, and the carbon chain of the fatty acid radical is substituted by two hydroxyl groups, with one hydroxyl group positioned along the carbon chain and one hydroxyl group positioned in the omega position thereof, or with two hydroxyl groups positioned along the carbon chain.

Use of glycolipid as an antimicrobial agent

The inventors have discovered that the glycolipid according to the invention has antimicrobial properties, in particular antibacterial properties.

The invention therefore also relates to the use of the glycolipid as defined above as an antimicrobial agent, in particular as an antibacterial agent.

In the sense according to the invention, the term “antimicrobial agent” designates a compound capable of destroying microbes (i.e. a microbiocidal agent) or slowing their growth (i.e. microbiostatic agent). By “microbe” we mean viruses or unicellular or multicellular microorganisms.

By way of example, the glycolipid according to the invention can be used as a preservative agent which inhibits the development of microorganisms and makes it possible to increase the shelf life of products, in particular in the cosmetic, pharmaceutical, or food.

Use of glycolipid as a surfactant

The inventors have also discovered that the glycolipid according to the invention can also be used as a surfactant, in particular as an emulsifying agent. The invention therefore also relates to the use of the glycolipid as defined above as a surfactant, in particular as an emulsifying agent.

In the sense according to the invention, a surfactant is a substance modifying the surface tension between two surfaces; an emulsifying agent is a surfactant which facilitates the formation of an emulsion, or improves its stability by reducing its rate of aggregation and/or coalescence.

In particular, the glycolipid according to the invention can be used as an emulsifier for the preparation of an oil-in-water emulsion.

In the sense according to the invention, an oil-in-water emulsion comprises an oily phase dispersed within an aqueous phase, and a water-in-oil emulsion comprises an aqueous phase dispersed within an oily phase.

The inventors have discovered that, surprisingly, when a glycolipid according to the invention is used as an emulsifying agent for the preparation of an oil-in-water emulsion, this emulsion is sensitive to pH, more particularly that it is kinetically stable as long as the pH of the aqueous phase of the emulsion is greater than a threshold hydrogen potential pH _s , and that it destabilizes when this pH is adjusted so as to be lower than the pH _s .

pH sensitive emulsion

Another object according to the invention is therefore an oil-in-water emulsion comprising an oily phase dispersed in an aqueous phase and at least one glycolipid of formula (I), characterized in that the emulsion is stable when the pH of the aqueous phase is greater than a threshold hydrogen potential pH _s .

In the present invention, the expression "kinetically stable as long as the pH of the aqueous phase of the emulsion is greater than a threshold hydrogen potential pH _s " means that the pH of the aqueous phase of the emulsion must be greater than pH _s so that the emulsion is stable. Advantageously, the pH _s corresponds to the pKa of the glycolipid according to the invention used as emulsifier. In the case where the emulsion comprises a mixture of glycolipids according to the invention, pH _s advantageously corresponds to the pKa of the mixture of glycolipids according to the invention.

Preferably, the threshold hydrogen potential pH _s ranges from 2 to 5, and preferably from 2 to 4, and more preferably is approximately 4.

When the pH of the aqueous phase of the emulsion is lower than the pH _s as described above, the emulsion destabilizes in a few minutes, typically 5 to 20 minutes, and is completely destroyed in a few hours, typically 2 at 24h.

In one embodiment, the emulsion comprises a mixture of glycolipids according to the invention as described above.

Without wishing to be bound by a particular theory, the inventors believe that the instability of the emulsions observed at low pH reflects the existence of a strong electrostatic component in the stabilization of the emulsions by the glycolipids according to the invention.

Nature and quantity of the oily and aqueous phases

Preferably, the aqueous phase advantageously represents at least 50% by weight of the emulsion, relative to the total weight of the emulsion. Preferably, the emulsion according to the invention comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% by weight of aqueous phase, relative to the total weight of the emulsion.

The quantity of aqueous phase in the emulsion according to the invention is notably between 50 to 80% by weight, preferably between 50 to 70% by weight, and more preferably between 50 to 60% by weight relative to the total weight of the emulsion.

Advantageously, the aqueous phase represents between 50 and 70% by weight of the emulsion. The aqueous phase of the emulsion according to the invention mainly comprises water, a glycolipid or a mixture of glycolipids according to the invention and optionally one or more compounds miscible with water.

Preferably, the aqueous phase comprises, in % by weight of aqueous phase, from 0.5 to 5%, preferably from 0.5 to 3%, more preferably from 1 to 3% of a glycolipid or a mixture of glycolipids according to the invention.

The aqueous phase may also include ionic species, pH regulators, active ingredients, preservatives, or even dyes, said active ingredients, preservatives and dyes being water-soluble or water-dispersible.

The oily phase of the emulsion according to the invention is a fatty phase comprising at least one fatty substance chosen from liquid fatty substances at room temperature (20-25°C) or oils, volatile or not, of vegetable or mineral origin. or synthetic, and mixtures thereof. By “oil” we mean a liquid fatty substance at room temperature (25°C). The oily phase may also include any liposoluble or lipodispersible additive.

For application in the cosmetic or pharmaceutical field, these oils are chosen from physiologically acceptable oils.

As oils which can be used in the emulsion according to the invention, mention may be made, for example, of hydrocarbon oils of animal origin; hydrocarbon oils of plant origin; synthetic esters and ethers, particularly of fatty acids; linear or branched hydrocarbons, of mineral or synthetic origin, such as paraffin oils, volatile or not, and their derivatives; partially hydrocarbon and/or silicone fluorinated oils; silicone oils; and their mixtures.

Among the hydrocarbons of the crude formula C _n H _m with n and m being two whole numbers, linear, aromatic and cyclic hydrocarbons can be used, whatever their boiling point. Mention will more particularly be made as hydrocarbons which may be present in the oily phase according to the invention: cyclopentane (C5H10), hexane (C6H14), methylcyclohexane (C7H14), heptane (C7H16), decane (C10H22), dodecane (C12H26), hexadecane (C16H34), and toluene (C7H8). The oily phase in the emulsion according to the invention may be composed of a single oil, in particular a single hydrocarbon, or may also consist of a mixture of two, three or even four different oils.

In the emulsion, the oily phase is typically in the form of drops of oily phase suspended in the aqueous phase.

The oil phase drops are preferably monodisperse.

The particle size distribution of the oil phase drops can be characterized in terms of average drop diameter in volume, D(4.3), and polydispersity in size, P, defined by:

where Ni is the number of drops of diameter Di, and D _m is the median diameter (diameter which divides the distribution into two parts of equal areas).

The measurement of the median diameter of the drops can be carried out from a size histogram obtained by measuring the individual diameters of an assembly of drops (minimum 500) on one or more images taken by optical microscopy or by measuring diffusion static of light.

The drops of oily phase advantageously have a volume average diameter of the order of a few micrometers. Thus, the droplets generally have a volume average diameter of between 2 and 10 pm, preferably between 3 and 6 pm, more preferably between 4 and 5 pm. Dry emulsion

The inventors have also shown that the emulsion according to the invention can be subjected to a treatment to produce a dry emulsion, the treatment consisting of eliminating the majority or all of the aqueous phase of the emulsion according to the invention by drying or freeze-drying.

An object according to the invention therefore relates to an emulsion according to the invention in the form of a dry emulsion.

In the sense according to the invention, a dry emulsion is in the form of a powder consisting of oily particles capable of restoring an oil-in-water emulsion after rehydration, that is to say after dispersion of the dry emulsion in an aqueous phase. The terms “emulsion in the form of a dry emulsion” and “dry emulsion” are used interchangeably.

Preferably, the dry emulsion comprises drops of oily phase, a glycolipid according to the invention and less than 20%, preferably less than 10% by weight of aqueous phase, relative to the total weight of the dry emulsion. Advantageously, the dry emulsion comprises at most 1, or 2% by weight of aqueous phase, relative to the total weight of the dry emulsion. Advantageously again, the dry emulsion is devoid of aqueous phase.

The oily phase drops have an average size of around 4 pm.

According to a preferred mode, the dry emulsions according to the invention contain a protective saccharide compound, in particular chosen from the group consisting of sucrose, lactose, fructose, trehalose, dextrins, maltodextrins, yellow dextrins, invert sugars, sorbitol, polydextrose, starch syrup, glucose syrup and mixtures thereof. Preferably, the protective saccharide compound is lactose.

The dry emulsion is advantageously obtained by freezing an emulsion according to the invention, typically at a temperature between -50°C and -100°C and lyophilizing it. Process for preparing an emulsion

The invention also relates to the preparation of an emulsion according to the invention, comprising: a) preparation of an aqueous phase and an oily phase, in which the oily phase and/or the aqueous phase comprises the glycolipid according to invention; b) combination of the aqueous phase and the oily phase and stirring until an emulsion is obtained.

Preferably, the oily phase and/or the aqueous phase comprises the glycolipid according to the invention in an aqueous phase/glycolipid mass ratio of between 60 and 10.

Preferably, the aqueous phase of step a) comprises, in % by weight of aqueous phase, from 0.5 to 5%, preferably from 0.5 to 3%, more preferably from 1 to 3% of glycolipid. according to the invention. In one embodiment, the oily phase does not comprise a glycolipid according to the invention.

During step a), the pH of the aqueous phase is preferably adjusted so as to be greater than the pKa of the glycolipid according to the invention.

The combination of the oily phase and the aqueous phase is done in the most appropriate order, as is known to those skilled in the art. In a particular embodiment, step b) is carried out by adding the oily phase to the aqueous phase while stirring the resulting combination.

Using the emulsion for cleaning surfaces

Another object according to the invention concerns the use of an emulsion according to the invention for cleaning surfaces.

Indeed, surprisingly, the inventors discovered that when a drop of emulsion according to the invention is deposited on a solid substrate, such as a glass slide, or a plastic support, it moves, s spreads and contracts spasmodically and almost periodically on the support. This gives the emulsion the ability to be self-spreading or even self-stirring, which is of interest in the field of surface cleaning. Without wanting to be bound by a particular theory, the inventors attribute these properties to convective phenomena taking place within the emulsion drop.

EXAMPLES

Enzymes studied, organisms of origin and specificity

The enzymes used in the Examples are listed in Table 1.

Table 1: GH70 a-transglucosylases, specificity of bonds during the synthesis of the natural polymer. P: polymerase ie glucan-sucrase GH70; B: branching sucrase GH70; nd= not determined The origin of the enzymes in Table 1 are listed in Table 2.

Table 2: Organisms of origin of GH70 a-transglucosylases

Heterologous expression of enzymes in Escherichia Coli

The genes coding for the enzymes mentioned above are cloned into vectors allowing recombinant expression in E. coli (see Table 3).

Recombinant enzymes are produced from cells of E. coli BL21 Star DE3 (SEQ ID NO 18, 13, 1, 3, 4, 5, 6, 16 and 17), BL21 Al DE3 (SEQ ID NO 12, 14, and 2) or Top 10 (SEQ ID NO: 15 ) transformed with the plasmid containing the gene for the targeted enzymes (see Table 3).

Table 3: Plasmids of the enzymes used and expression systems adapted to the different enzymes. (Gly: Glycerol; Glu: Glucose; a-Lac: a-Lactose; L-Ara: L- Arabinose)

Three hundred microliters of the transformation mixture make it possible to inoculate a volume of 30 mL of LB (Lysogeny Broth), supplemented with 100 pg.mL' ¹ of ampicilin. The medium is incubated overnight at 37°C in order to prepare a preculture. 1 L cultures in modified ZYM5052 medium (Studier, 2005) whose characteristics are presented in Table 3 are inoculated at an initial ODx=6oonm of 0.05 from the preculture of the day before, then incubated 26 hours at 21°C and 150 rpm. At the end of fermentation, the culture media are centrifuged (15 min, 6500 rpm, 4°C) and the pellets are concentrated to a ODx=6oonm of 80 in activity buffer (see Table 3). The cells are then broken with ultrasound according to the following protocol: 5 cycles of 20 seconds at 30% of the maximum power of the probe, cold, spaced by 4 minutes of rest on ice. The sonication supernatants containing the soluble enzymes of interest are then recovered after 30 minutes of centrifugation (10,000 rpm, 10°C) and stored at 4°C.

Determination of enzyme activity by assay of reducing sugars in DNS.

Enzymatic activity is determined by measuring the initial rate of production of reducing sugars using the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959).

An enzyme unit represents the quantity of enzyme which releases one pmole of fructose per minute, at 30°C, from 100 ^g.L'1 of sucrose in 50 mM sodium acetate buffer, pH 5.75. The activity is determined by measuring the initial rate of production of the reducing sugars using the 3,5-dinitrosalicylic acid (DNS) method.

During kinetics, 100 μL of reaction medium are taken and the reaction is stopped by adding an equivalent volume of DNS. The samples are then heated for 5 min at 95°C, cooled in ice, diluted one-half in water, and the absorbance is read at 540 nm. A standard range of 0 to 2 ^g.L'1 of fructose makes it possible to establish the link between absorbance value and reducing sugar concentration.

Example 1: Screening of enzymes in acceptor reaction with different hydroxy fatty acids

1.1. Material and methods

Reaction conditions

The acceptor reactions are carried out in conical microtubes, in a volume of 1 mL. The screening of GH family 70 enzymes was carried out under the following conditions:

- [sucrose]o = 292 mM (100 g.L' ¹ )

- [hydroxylated fatty acid]o = 10 mM, - 10% v/v DMSO

- 50 mM sodium acetate buffer at pH=5.75

- enzymatic activity: 1 U.mL' ¹

- temperature: 30°C

- agitation: 800 rpm

The hydroxy fatty acid is initially dissolved in 100% DMSO.

The hydroxylated fatty acids (hereinafter AGH) tested are respectively: a sulfanyl hydroxylated fatty acid: 11-[2-[hydroxy-alkyl)-sulfanyl]-undecanoic acid (hereinafter AHESll) a co-hydroxylated fatty acid: acid 11 hydroxyundecanoic acid (hereinafter AHIID) a poly-hydroxy fatty acid: erythro-aleuritic acid (hereinafter AEA)

The final concentration of DMSO in the reaction medium is 10% (v/v). The reaction is initiated by the addition of a volume of cell lysate sufficient to obtain an enzymatic activity in reaction of 1 ^U.mL'1 . The reactions are incubated at 30°C and stirred at 800 rpm using an Eppendorf ThermoMixer C. After 24 hours, the enzymes were denatured at 95°C for 5 min.

Analytical techniques

For their analyzes by HPLC, the reaction media are diluted one-half in absolute ethanol. This dilution allows, among other things, the elimination by precipitation of potential high molecular mass polymers.

The separation of the lipid acceptors and their glucosylated forms is carried out in reverse phase with a Synergi™ Fusion-RP column (porosity of 80 Å, particle size of 4 μm, C18 grafting with polar termination, Phenomenex, USA). This column is maintained at 30°C on a Thermo U3000 HPLC system coupled to a Corona CAD Véo detector (Charged Aerosol Detector) (Thermo Scientific, USA). The nebulization temperature is set at 50°C and the filter maintained at 3.2 seconds.

The mobile phase is composed of a mixture of ultrapure water (solvent A) / HPLC quality acetonitrile (solvent B) each containing 0.05% (v/v) formic acid. The elution is carried out at a flow rate of 1 mL.min' ¹ according to the following gradient: Between 0 min and 5 min, a first elution phase at 0% (v/v) of route B allows the elimination of residual simple sugars (fructose, glucose, leucrose, and residual sucrose or possible short oligosaccharides),

A second isocratic elution phase at a percentage of 35% of route B is carried out for 25 minutes to separate the different glucosylated lipid compounds,

A final phase of 10 minutes at 75% of channel B allows the regeneration of the column.

Acceptor conversion rate

The ability of enzymes to glucosylate an acceptor, here AHESU, AHUD or AEA, is determined by measuring the conversion rate of the acceptor between the start and the end of the reaction. The conversion rate is calculated as follows:

[Math. 1]

where Acceptor is the conversion rate of the acceptor, [Acceptor^ its initial concentration and [Acceptor^ its final concentration.

1.2. Results

Glucosylation of AHESU

Figure 1 illustrates the ability of the enzymes to glucosylate AHESU under the conditions set out above.

The GH family 70 enzymes tested were found to be active on AHESU.

The branching enzymes BRS-A, BRS-B A1, BRS-B, BRS-E A1 and GBD-CD2 AN123 were found to be particularly active. Indeed, conversion rates ranging from 20% to rates greater than 80% under the screen conditions tested were obtained with these enzymes. Glucosylation of AHIID

Figure 2 illustrates the ability of the enzymes to glucosylate AHUD under the conditions set out above.

All GH family 70 enzymes were found to be active on AHIID.

The branching enzymes BRS-A, BRS-B A1, BRS-B, BRS-E A1 and GBD-CD2 AN123 were found to be particularly active. Indeed, conversion rates ranging from 18% to rates greater than 60% under the screen conditions tested were obtained with these enzymes.

Glucosylation of IAEA

Figure 3 illustrates the ability of the enzymes to glucosylate AEA under the conditions set out above.

All GH family 70 enzymes were found to be active on AEA.

The branching enzymes BRS-A, BRS-B A1, BRS-B, BRS-E A1 and GBD-CD2 AN123 were found to be particularly active. Indeed, conversion rates ranging from 20% to rates greater than 60% under the screen conditions tested were obtained with these enzymes.

Example 2: Comparison of the profiles of glucosylation products of AHESU, AHUD and AEA by the BRS-A branching enzymes; BRS-B A1; BRS-C; BRS-D A1, BRS-E A1 and GBD-CD2 AN 123

2.1. Material and methods

The reactions are carried out at 30°C with 800 rpm stirring in the presence of 438.5 mM sucrose, 10 mM AGH (AHESU, AHUD or AEA), 10% v/v DMSO and sodium acetate buffer. 50 mM at pH=5.75 and BRS-A; BRS-B A1; BRS-C; BRS-D A1, BRS-E A1 or GBD-CD2 AN123 (enzymatic activity of 1 ^U.mL'1 ). 2.2. Results

2.2.1. AHESll qlucosylation product profiles

Figure 4 illustrates the diversity of glucosylation products obtained under the conditions set out above.

The glucosylation products are eluted between 10 and 16 minutes of the HPLC analysis. The product detected at 26.5 minutes corresponding to the residual AHESU acid.

For each enzyme tested, the chromatographic profiles show numerous glucosylation peaks. For example, six major products are obtained when BRS-A catalyzes the reaction.

The profile of glucosylation products depends on the enzyme considered.

The majority glucosylation products seem more numerous with the branching enzymes BRS-A, BRS-D A1 and GBD-CD2 AN123 specific for the a-1,2 bond, particularly in the products eluted in the early stages of the analysis. Indeed, three to four high intensity peaks are observable with these enzymes.

Conversely, only two main products are obtained with BRS-B A1, BRS-C and BRS-EA1.

With the exception of BRS-C and BRS-E A1 enzymes, the glucosylation efficiency is very high with AHESU as acceptor, particularly for BRS-A, BRS-B A1, BRS-D A1, and GBD- CD2 AN123. Indeed, the conversion rates of AHESU obtained with 438.5 mM (150 ^g.L'1 ) of sucrose are 94%, 84% and 75% for BRS-A, BRS-D A1, and GBD-CD2 AN 123 respectively (BRS specific for the a-1,2 bond) and 89%, 26%, 22% for BRS-B A1, BRS-C and BRS-E A1 (BRS specific for the a-1,3 bond).

2.2.2. AHUD Glucosylation Product Profiles

Figure 5 illustrates the diversity of glucosylation products obtained under the conditions set out above. The glucosylation products obtained are eluted between 10 and 25 minutes. The product detected at 49 minutes corresponding to residual 11-hydroxyundecanoic acid (AHUD). The product profiles obtained are complex with numerous glucosylation peaks detected (a dozen different products for BRS-A for example).

The profile of glucosylation products is also dependent on the enzyme considered. The majority products Nos. 1 and 2 are, for example, in varying proportions depending on the enzymes: a high production of glucosylation product No. 1 is observed with GBD-CD2 AN 123; on the contrary, a production of the same order of magnitude of the two glucosylation products (1 and 2) is obtained with BRS-E A1.

The products eluted in the first moments of the analysis (between 10 and 15 minutes) appear in greater quantities in the reactions catalyzed by enzymes specific to the a-1,2 bond, ie. BRS-A, BRS-D A1 and GBD-CD2 AN 123.

The efficiency of glucosylation is also dependent on the enzyme considered.

Indeed, the AHUD conversion rates obtained are 78%, 40%, 17%, 54%, 32% and 67% for BRS-A, BRS-B A1, BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN123 respectively under the conditions shown in Figure 5.

2.2.3. AEA Glucosylation Product Profiles

Figure 6 illustrates the diversity of glucosylation products obtained under the conditions set out above.

The glucosylation products obtained are eluted between 10 and 30 minutes of the HPLC analysis, the product detected at 43 minutes corresponding to the residual AEA. The glucosylation product profiles appear complex, with numerous glucosylation peaks being detected (more than 16 major products for BRS-A for example).

The profile of glucosylation products is very dependent on the enzyme considered. Indeed, if peak 4 is common to all enzymes, peak 5 is characteristic of the BRS-B A1 enzyme. Peak 10 is essentially present in the products obtained with the enzymes BRS-A and BRS-D A1, two enzymes specific for the α-1,2 bond. The products of glucosylation 1 to 3 are essentially produced by BRS-B A1 and BRS-E A1, two enzymes specific for the a-1,3 bond.

The efficiency of glucosylation is also dependent on the enzyme considered. Indeed, the AEA conversion rates obtained are 63%, 74%, 30%, 60%, 65% and 74% for BRS-A, BRS-B A1, BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN123 respectively under the conditions shown in Figure 6.

Example 3: Effect of the initial concentration of sucrose on the profile of the glucosylation products of AHESU, AHUD and AEA obtained with BRS-A; BRS-B A1; BRS-C; BRS-D A1, BRS-E A1 or GBD-CD2 AN123.

3.1. Material and methods

Glucosylation Product Profile

The reactions are carried out at 30°C with 800 rpm stirring in the presence of 146 mM, 292 mM, 439 mM, 585 mM or 731 mM sucrose; 10 mM AGH (AHESU, AHUD or AEA); 10% v/v DMSO and 50 mM sodium acetate buffer at pH=5.75 in the presence of BRS-A; BRS-B A1; BRS-C; BRS-D A1, BRS-E A1 or GBD-CD2 AN123 (Enzymatic activity 1 U.mL' ¹ , T=30°C)

3.2. Results

3.2.1. Effect of initial sucrose concentration on the conversion rate and profile of AHESU glucosylation products

Increasing the initial sucrose concentration has the effect of optimizing the AHESU conversion rate (Figure 7). This effect is particularly marked for the enzymes BRS-A, BRS-B A1, BRS-D A1 and GBD-CD2 AN123. Indeed, the conversion rate of between 25% and 55%, obtained at an initial concentration of 146 mM, increases to a rate greater than 85% (up to 98% for BRS-A).

Increasing initial sucrose concentrations also have an effect on the profiles of glucosylation products (not shown). For example, for the four enzymes BRS-A, BRS-B A1, BRS-D A1 and GBD-CD2 AN123, the eluted glucosylation products in the first moments of the analysis (certainly products with a higher degree of glucosylation) are clearly favored by the increase in the initial sucrose concentration.

3.2.2. Effect of initial sucrose concentration on the conversion rate and profile of AHUD glucosylation products

Increasing the initial sucrose concentration has the effect of optimizing the AHUD conversion rate (Figure 8).

This effect is mainly marked for enzymes with a-1,2 specificity. Indeed, the conversion rate of between 25% and 55%, obtained at an initial concentration of 146 mM with BRS-A, BRS-D A1 and GBD-CD2 AN 123 increases to a rate of between 60% and 85 % for a concentration greater than 146 mM. This effect of the initial sucrose concentration is less marked for BSR-B A1, BSR-C and BSR-E A1.

In comparison, the conversion rate obtained with enzymes of a-1,3 specificity does not exceed 40% for an initial sucrose concentration of 730 mM.

Increasing initial sucrose concentrations also have an effect on the overall profile of glucosylation products (not shown). For example, for the three branching enzymes BRS-A, BRS-D A1 and GBD-CD2 AN 123, the glucosylation products eluted in the first moments of the analysis are clearly favored by the increase in the initial sucrose concentration. . These products are certainly products with a higher degree of glucosylation. In the case of a-1,3 branching enzymes, this effect is much less marked.

3.2.3. Effect of initial sucrose concentration on the conversion rate and profile of AEA glucosylation products

Increasing the initial sucrose concentration has the effect of reducing the residual AEA peak. The conversion of the acceptor is therefore optimized. Indeed, the conversion rate of between 37 and 50% obtained at an initial concentration of 146 mM increases to a rate of between 76% and 85% depending on the enzymes considered (BRS-B A1, BRS-D A1, BRS -E A1 and GBD-CD2 AN123). This effect of concentration initial sucrose is less marked for the BRS-C enzyme. This AEA conversion optimization is illustrated in Figure 9.

Increasing initial sucrose concentrations also have an effect on the overall profile of glucosylation products (not shown). For example, for the two branching enzymes BRS-A and BRS-D A1 (which glucosylate dextran into α-1,2), the glucosylation products eluted in the first moments of the analysis are clearly favored with the increase of the initial concentration of sucrose. These products are certainly products with higher degrees of glucosylation.

In the case of a-1,3 branching enzymes, the synthesis of glucosylation product no. 4 is disadvantaged in favor of that of other glucosylation products, especially glucosylation product no. 5 in the case of the BRS enzyme -B.

Example 4: Structural characterization of the main glucosylation products of AHESU by BRS-A, of AHUD by BSR-B A1 and of AEA by BSR-B A1

4.1. Production and structural characterization of AHESU glucosylation products

4.1.1. Production of a batch of AHESU glycolipids at gram scale

Glucosylation of AHESU is catalyzed by the BRS-A enzyme on 500 mg of AHESU. The reaction conditions are as follows:

- [sucrose]o = 438.5 mM

- [AHESU]o = 10 mM (initially dissolved in DMSO at 100 mM)

- sodium acetate buffer at 50mM and pH = 5.75

- ultra-pure water qsp 200mL

- activity of BRS-A in reaction: 1 U.mL' ¹

The reaction is carried out at 37°C with magnetic stirring. After 24 hours, the reaction is stopped by incubation at 95°C for 10 minutes. The mixture is stored at -20°C before purification.

A prepurification step of the glucosylated hydroxy-lipids from AHESU is carried out by flash chromatography using a column containing 250 mL of stationary phase. Purosorb PAD910, Purolite, USA. The glucosylation products are thus separated from the residual free sugars using the following elution steps:

- 4 column volumes (VC) of ultra-pure water until complete elution of residual sugars

- Elution of glycosylation products using 4 VC of an l-O/Ethanol 35% / 65% v/v mixture.

- Return to 100% ultra pure H2O in 5 VC.

The glucosylation products are collected and concentrated by rotary evaporation under vacuum then analyzed by chromatography. The chromatogram obtained at this prepurification step is presented in Figure 10. There are at least 6 main glucosylation products noted in the reverse order of their polarity. Product no. 1 corresponds to that eluted closest to AHESU, a lipid acceptor that has not completely reacted.

The majority glucosylated forms of AHESU (glucosylation products no. 1 to 3) are isolated using a semi-preparative system consisting of an Agilent 1260 Infinite HPLC chain coupled to a “Dionex Ultimate 3000 automated fraction collector” . Separation is ensured by a Synergi™ Fusion-RP column (porosity of 80 Å, particle size of 4 μm, C18 grafting with polar termination, Phenomenex, USA). The elution is carried out isocratically with a mixture of H2O/Acetonitrile 65%/35% v/v at a flow rate of 1 mL.min' ¹ .

4.1.2. Characterization of AHESU glucosylation products a) High resolution mass spectrum (LTQ Orbitrap) of AHESU glucosylation products

In order to determine the exact masses of the purified glucosylation products, the Thermo U3000 HPLC system was coupled to a high-resolution LTQ Orbitrap Vélos spectrometer (ThermoScientific, USA). Ionization is carried out in negative electrospray mode (H ESI II -) with a mass delta of +/- 5 ppm. The high-resolution mass spectra of glucosylation products #1 to 3 are obtained (Figure 10). High resolution LC-MS analysis of the glucosylation product of AHESU n°1 whose retention time is 7.8 min leads to the identification of a majority ion at m/z 423.20492 for [MH] ' corresponding to a mono-glucosylated form of AHESU.

A majority ion obtained with glucosylation product no. 2 (elution time = 6.5 minutes) at m/z 585.25716 for [M-H]' corresponds to a diglucosylated form of AHESU.

Surprisingly, glucosylation product no. 3 (peak no. 3 at 5.92 min) gives a main ion at m/z 585.2571 for ([M-H]'). Compound no. 3 is therefore also a diglucosylated form of AHESU but with a different sugar bond from that of product no. 2. b) NMR analysis of AHESU glucosylation products no. 1 to 3

The characterization of the glucosylation products of AHESU No. 1 (monoglucosylated AHESU), No. 2 and 3 (diglucosylated AHESU) is carried out by NMR. The ¹ H, ¹³ C, JMod, HSQC and HMBC spectra were recorded on Bruker Avance 500 MHz equipment at 298K with a BBI probe H-BB-D Z-Grd 5mm. The data were acquired and processed using TopSpin 3 software.

NMR characterization of AHESU's #1 glucosylation product

A ¹ H spectrum and a ¹³ C spectrum of the glucosylation product no. 1 of AHESU are produced.

The ¹ H and ¹³ C spectra obtained with glucosylation product no. 1 are in agreement with monoglucosylation of AHESU.

Peak No. 1 of the chromatograms presented in Figure 10 corresponds to 11-[(2-O-g-D-glucopyranosyl-ethyl)-sulfanyl]-undecanoic acid.

NMR characterization of AHESU glucosylation product no. 2

A ¹ H spectrum and a ¹³ C spectrum of the glucosylation product no. 2 of AHESU are produced. The presence of two anomeric protons at chemical shifts 4.84 and 5.13 ppm and two negative carbons at chemical shifts 100.54 ppm and 101.94 ppm (anomeric carbons) are consistent with a diglucosylated form of the lipid acceptor.

A 2D HSQC spectrum of AHESU glucosylation product no. 2 allows the assignment of anomeric protons and carbons for each sugar unit.

A 2D HMBC NMR spectrum of AHESU glycosylation product no. 2 shows an a-1,3 type saccharide bond between the two glucosyl units.

Peak No. 2 of the chromatograms presented in Figure 10 therefore corresponds to 11- [(2-O-a-D-glucopyranosyl-(1,3)-a-D-glucopyranosyl-ethyl) sulfanyl]-undecanoic acid.

NMR characterization of AHESU glucosylation product no. 3

A ¹ H spectrum and a ¹³ C spectrum of the glucosylation product no. 3 of AHESU are produced. The presence of two anomeric protons at chemical shifts 4.99 and 5.05 ppm and two negative carbons at chemical shifts 97.56 ppm and 98.28 ppm (anomeric carbons) are consistent with a second diglucosylated form of the acceptor lipid.

A 2D HSQC spectrum of AHESU glucosylation product no. 3 allows the assignment of anomeric protons and carbons for each sugar unit. A 2D HMBC spectrum of glucosylation product no. 3 shows an a-1,2 type saccharide bond between the two glucosyl units.

Peak No. 3 of the chromatogram presented in Figure 10 therefore corresponds to 11-[(2- O-a-D-glucopyranosyl-(1,2)-a-D-glucopyranosyl-ethyl) sulfanyl]-undecanoic acid.

4.2. Production and structural characterization of AHUD glucosylation products

4.2.1. Production of a batch of AHUD glycolipids on a gram scale.

The production of glucosylation products is carried out by the BRS-B enzyme on 1 g of AHUD. The reaction conditions are as follows:

- final sucrose concentration: 438.5 mM

- final AHUD concentration: 10 mM (initially dissolved in DMSO at 100 mM)

- sodium acetate buffer at 50mM and pH = 5.75 - ultra-pure water qsp 500 ml_

- activity of BRS-B in reaction: 1 U.mL' ¹

The reaction is carried out at 30°C with magnetic stirring. After 24 hours, the reaction is stopped by incubation at 95°C for 10 minutes. The mixture is stored at -20°C before purification.

4.2.2. Purification of AHUD glycolipids produced by the BRS-B enzyme.

A prepurification step of the glycolipids from the AHUD is carried out by flash chromatography using a REVELERIS® X2 Flash Chromatography System (GRACE, USA) equipped with a column containing 80 g of silica-C18 stationary phase. The glucosylation products are separated from the residual free sugars under the following conditions:

- flow rate of 60 ml. min ^-1

- 3 column volumes (VC) at 100% H2O

- gradient going from 100% ultra pure H2O to 100% acetonitrile in 2 VC

- 1 VC 100% acetonitrile

- return to 100% ultra pure H2O in 0.5 VC

- 100% ultra pure H2O equilibration for 1.5 VC

The peak corresponding to the glucosylation products is collected during the acetonitrile gradient. The chromatogram obtained during this pre-purification step by flash chromatography is presented in Figure 11.

LC-MS analysis in negative electrospray mode of the glucosylation product of AHUD No. 1 (retention time of 15.34 min) gives two majority ions at m/z 363.2 for [M-H]' corresponding to the mono-glucosylated form and m/z 725.3 for [2M-3H]' corresponding to a dimer.

LC-MS analysis in negative electrospray mode of the glucosylation product of AHUD No. 2 (retention time of 14.12 min) gives a majority ion at m/z 525.3 for [M-H]' corresponding to the di-glucosylated form.

The different glucosylated forms of AHUD are then purified on a semi-preparative system consisting of an Agilent 1260 Infinite CHLP chain coupled to a collector of fraction Dionex Ultimate 3000 automated fraction collector. Separation is ensured by a Synergi™ Fusion-RP column (porosity of 80 Å, particle size of 4 μm, C18 grafting with polar termination, Phenomenex, USA). The elution is carried out isocratically with a FW/Acetonitrile mixture 75%/25% v/v at a flow rate of 1 mL.min' ¹ .

4.2.3. NMR analysis of purified AHUD glycolipids.

The characterization of the glucosylation products of AHUD No. 1 (monoglucosylated AHUD) and No. 2 (diglucosylated AHUD) is carried out by NMR. The 1H, 13C, JMod, HSQC and HMBC spectra were recorded on Bruker Avance 500 MHz equipment at 298 K with a BBI probe H-BB-D Z-Grd 5mm. The data were acquired and processed using TopSpin 3 software.

NMR characterization of AHUD's #1 glucosylation product

A ¹ H spectrum and a ¹³ C spectrum of the glucosylation product no. 1 of AHUD are produced.

The ¹ H and ¹³ C spectra obtained with glucosylation product no. 1 of AHUD are in agreement with monoglucosylation of AHUD.

Peak No. 1 of the chromatogram presented in Figure 11 corresponds to 11-O-(a-D-glucopyranosyl)-undecanoic acid.

4.3. Production and structural characterization of AEA glucosylation products by BRS-B A1

4.3.1. Production of batches of glycosylated derivatives of erythroaleuritic acid on a gram scale.

The production of AEA glucosylation products is carried out by the BRS-B A1 enzyme on 1.5 g of AEA. The reaction conditions are as follows:

- Final sucrose concentration: 584.7 mM

- Final AEA concentration: 10 mM (initially dissolved in DMSO at 100 mM)

- Sodium acetate buffer at 50mM and pH = 5.75

- Ultra-pure water qsp 500 mL

- Activity of BRS-B in reaction: 1 U.mL' ¹ The reaction is carried out at 30°C with magnetic stirring. After 24 hours, the reaction is stopped by incubation at 95°C for 10 minutes. The mixture is stored at -20°C before purification.

4.3.2. Purification of glycolipids from AEA produced by the BRS-B A1 enzyme.

A prepurification step of the glycolipids from AEA is carried out by flash chromatography using a REVELERIS® X2 Flash Chromatography System (GRACE, USA) equipped with a column containing 80 g of Silica-Ci 8 stationary phase. glucosylation are separated from residual free sugars using the following elution steps at a flow rate of 60 ml. min ^-1 :

- 3 column volumes (VC) of 100% ultra pure F O.

- Gradient going from 100% ultra pure H2O to 100% acetonitrile in 2 VC.

- 1 VC 100% acetonitrile.

- Return to 100% ultra pure H2O in 0.5 VC

- Re-equilibration at 100% ultra pure H2O for 1.5 VC

Glucosylation products are collected during the acetonitrile gradient.

Figure 12 illustrates the chromatogram obtained during this pre-purification step by flash chromatography.

The LC-MS analysis in negative electrospray mode of the glucosylation product of AEA no. 4, the retention time of which is 16.16 min, leads to two majority ions at m/z 465.4 [M-H]' and m/z 579.3 [M+TFA-H]'. These ions confirm the obtaining of a mono-glucosylated form of AEA.

The LC-MS analysis in negative electrospray mode of the glucosylation product AEA n°5 whose retention time is 14.12 min leads to two majority ions at m/z 627.4 [M-H]' and m/z 741.4 [M+TFA-H]'). These two ions at +162 amu compared to peak 4 (m/z 465.4), confirm the obtaining of a di-glucosylated form of AEA.

These two main glucosylated forms of eryhtro-aleuritic acid are then purified on a semi-preparative system consisting of an Agilent 1260 Infinite HPLC chain coupled to a “Dionex Ultimate 3000 automated fraction collector”. Separation is ensured by a Synergi™ Fusion-RP column (porosity of 80 Å, particle size of 4 pm, C18 grafting with polar termination, Phenomenex, USA). The elution is carried out isocratically with a FW/Acetonitrile 78%/22% v/v mixture at a flow rate of 1 mL.min' ¹ .

4.3.3. NMR analysis of purified AEA qlucosylation products.

The characterization of the glucosylation products of AEA no. 4 (monoglucosylated AEA) and no. 5 (diglucosylated AEA) is carried out by NMR. The 1H, 13C, HSQC and HMBC spectra were recorded on Bruker Avance 500 MHz equipment at 298K with a BBI probe H-BB-D Z-Grd 5mm. The data were acquired and processed using TopSpin 3 software.

Characterization of AEA glycosylation product no. 4 (monoglucosylated AEA)

A ¹ H spectrum and a ¹³ C spectrum of the glucosylation product no. 4 of AEA are produced.

The ¹ H and ¹³ C spectra obtained with glucosylation product no. 4 of AEA are in agreement with monoglucosylation of AEA.

The ¹ H spectrum highlights an anomeric proton at a chemical shift of 4.77 ppm. The ¹³ C spectrum highlights the presence of negative carbon peaks C9 and C10 superimposed at a chemical shift of 75.38 ppm, which proves the absence of glucosylation on the two secondary hydroxyls. Glucosylation is therefore found at the level of positive carbon no. 16 deshielded at 69.21 ppm.

Peak No. 4 of the chromatogram presented in Figure 12 corresponds to 16-O-(a-D-glucopyranosyl)-9,10-dihydroxy-hexadecanoic acid.

Characterization of AEA glucosylation product no. 5 (diglucosylated AEA)

A ¹ H spectrum and a ¹³ C spectrum of the AEA glucosylation product no. 5 are produced.

The ¹ H and ¹³ C spectra obtained with AEA glucosylation product no. 4 are in agreement with diglucosylation of AEA.

The ¹ H spectrum highlights two anomeric protons at a chemical shift of 4.79 ppm and 5.14 ppm and two anomeric carbons at 100.34 ppm and 101.89 ppm. Like the monoglucosylated form, the negative carbon peaks C9 and C10 are still present at 75.41 ppm, which demonstrates diglucosylation at position C16.

The 2D HMBC spectrum of AEA shows an a-1,3 type saccharide bond between the two glucosyl units.

Peak No. 5 of the chromatogram presented in Figure 12 corresponds to a diglucosylated AEA of the 16-O-(a-D-glucopyranosyl)-(1,3)-a-D-glucopyranosyl)-9,10-dihydroxy-hexadecanoic acid type.

Example 5: Glucosylation of omega-hydroxy fatty acids having a hydrocarbon chain comprising a carbon number greater than 11

The so-called branching enzymes BRS-A, BRS-B A1, BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN 123 were tested to glucosylate 12-HydroxyDoDecanoic Acids (AHDD), 15-HydroxyPentaDecanoic Acids (AHPD) and 16-HydroxyHexaHecanoic (AHHD) comprising 12, 15 and 16 carbon atoms respectively.

Figure 13 shows the glucosylation products obtained at 37°C with 800 rpm stirring under the following synthesis conditions:

- Initial concentration of sucrose: 877 mM

- Initial concentration of omega-hydroxy acid: [AHDD]=10mM; [AHPD]=1 mM and [AHHD]=0.5 mM,

- 10% v/v DMSO

- 50 mM sodium acetate buffer at pH=5.75

- Enzymatic activity: 1 U.mL' ¹

For analysis by HPLC, the reaction media are diluted one-half in absolute ethanol. The separation is carried out with a Synergi™ Fusion-RP column (porosity of 80 Å, particle size of 4 μm, C18 grafting with polar termination, Phenomenex, USA). The elution is carried out at 30°C on a Thermo U3000 HPLC system coupled to a Corona CAD Véo detector (Charged Aerosol Detector) (Thermo Scientific, USA). The nebulization temperature is set at 50°C and the filter maintained at 3.2 seconds. The mobile phase is composed of a mixture of ultrapure water (solvent A) / HPLC quality acetonitrile (solvent B) containing 0.05% (v/v) formic acid. The elution is carried out at a flow rate of 1 mL.min' ¹ according to the following gradient:

- Between 0 min and 5 min, a first elution phase at 0% (v/v) of route B.

- A gradient phase going from 0% of channel B to 100% of channel B in 35 minutes.

The analysis of the two chromatograms presented in Figure 14 proves the possibility of glucosylation of long-chain fatty acids carrying primary hydroxyl in the œ position (up to C16 at least).

Indeed, the comparison of the initial (control) and final reaction times shows the appearance of numerous glucosylation products between elution times 17 minutes and 22 minutes for AHDD, between 19 minutes and 25 minutes for AHPD. and between 21 minutes and 27 minutes for AHPD.

The glucosylation efficiency is dependent on the chain size of the co-hydroxylated acceptors considered. Glucosylation of acceptors of higher chain length is obtained only by decreasing the working concentrations (from 10 mM for AHDD to 0.5 mM for AHHD for example).

Glucosylation of 12-hvdroxydodecanoic acid (AHDD)

The branching enzymes BRS-A, BRS-D A1, GBD-CD2 AN123 (specific for the α-1,2 bond) and the BRS-E enzyme (specific for the α-1,3 bond) appear in this case still be the most effective enzymes.

In fact, the conversion rates for AHDD are 60%; 40% and 42% for enzymes. BRS-A, BRS-D A1 and GBD-CD2 AN 123 respectively and only 18%; 11% and 17% for the enzymes BRS-B A1, BRS-C and BRS-E A1 respectively.

The chromatograms obtained show multi-glucosylation profiles with numerous peaks, two of which are predominantly eluted at 21 and 22 minutes. Glucosylation of 15-hydroxypentadecanoic acid (AHPD) and 16-hydroxyhexadecanoic acid (AHHD)

Glucosylation of longer chain acceptors is more efficient with the two branching enzymes BRS-A (a-1,2 specific) and BRS-B (a-1,3 specific). In fact, the conversion rates obtained with these acceptors are:

- for the AHPD, 70% for BRS-A and 33% for BRS-C. The transformation yields of AHPD are only 5.0%, 22%, 13%, 29% for BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN 123 respectively.

For AHHD, 38% for BRS-A and 21% for BRS-C. The transformation yields of AHHD are only 5%, 12%, 6% and 12% for BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN 123 respectively.

Example 6: Glucosylation of 9,10-dihydroxyoctadecanoic and 9,10,12-trihydroxyoctadecanoic acids by the enzymes BRS-A, BRS-B A1, BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN 123

The enzymes BRS-A, BRS-B A1, BRS-C, BRS-D A1, BRS-E A1 and GBD-CD2 AN123 (a-branching transglucosylase) were tested to glucosylate 9,10-dihydroxyoctadecanoic acids (ADHO ) and 9, 10, 12-trihydroxyoctadecanoic acid (ATHO).

Figure 14 shows the glucosylation products obtained at 37°C with 800 rpm stirring under the following synthesis conditions:

- Initial concentration of sucrose: 877 mM

- Initial concentration of poly-hydroxy fatty acid (ADHO and ATHO): 5 mM, - 10% v/v of DMSO

- 50 mM sodium acetate buffer at pH=5.75

- Enzymatic activity: 1 U.mL' ¹

For analysis by HPLC, the reaction media are diluted one-half in absolute ethanol. The separation is carried out with a Synergi™ Fusion-RP column (porosity of 80 Å, particle size of 4 m, C18 grafting with polar termination, Phenomenex, USA). The elution is carried out at 30°C on a Thermo U3000 HPLC system coupled to a Corona CAD Véo detector (Charged Aerosol Detector) (Thermo Scientific, USA). The nebulization temperature is set at 50°C and the filter maintained at 3.2 seconds. The mobile phase is composed of a mixture of ultrapure water (solvent A) / HPLC grade acetonitrile (solvent B) containing 0.05% (v/v) formic acid. The elution is carried out at a flow rate of 1 mL.min' ¹ according to the following gradient:

- Between 0 min and 5 min, a first elution phase at 0% (v/v) of route B.

- A gradient phase going from 0% of channel B to 100% of channel B in 30 minutes.

The analysis of the two chromatograms presented in Figure 14A and Figure 14B proves the possibility of glucosylation of fatty acids carrying secondary hydroxyl present within the carbon chain only.

Indeed, the comparison of the initial (control) and final times of the reactions shows the appearance of numerous glucosylation products between the elution times 21 minutes and 26 minutes for ADHO and 19 minutes and 23 minutes for ATHO. The formation of these glucosylation products appears much more efficient with the trihydroxylated fatty acid (ATHO) than with the dihydroxylated form ADHO, the intensity of the peaks observed being significantly greater in the first case (ATHO).

Likewise, the conversion rates vary between 2% and 30% for ATHO and between 1% and 11% for ADHO depending on the branching enzymes considered.

The branching enzymes BRS-A, BRS-D A1, GBD-CD2 AN123 (specific for the α-1,2 linkage) and the enzyme BRS-E (specific for the α-1,3 linkage) appear to be the enzymes most effective in both cases.

Example 7: Glucosylation of hydroxylated fatty acids by mutants of the GBD CD2 AN123 enzyme

Mutants of the GBD CD2 AN 123 enzyme were tested to glucosylate the following hydroxy fatty acids: 11-[2-[hydroxy-alkyl)-sulfanyl]-undecanoic acid (AHESU), 11-hydroxyundecanoic acid (AHUD), 12-acid -hydroxydodecanoic acid (AHDD), 15-hydroxypentanoic acid (AHPD), erythro-aleuritic acid (AEA), 9,10,12-trihydroxy octadecanoic acid (ATHO). The results show that these mutants are very efficient insofar as they have much higher conversion rates. Conversion rates of the following hydroxy fatty acids are obtained:

Conversion rates obtained with GBD CD2 AN 123 mutants.

The GBD-CD2 AN 123 mutants also make it possible to obtain glucosylation products having larger saccharide units.

Example 8: Study of the emulsifying properties of the glucosylation products of AHESU by BRS-A and of AHUD by BRS-B A1

8.1. Preparation of emulsions

The emulsifying properties of the glucosylation products of AHESU by the enzyme BRS-A (hereinafter referred to as Glc-AHESU) and of AHUD by BRS-B A1 (hereinafter Glc-AHUD) were evaluated. Aqueous phase

An aqueous solution comprising 2% by weight of Glc-AHESU is prepared.

The pH of the aqueous solution is adjusted using a 1 M NaOH solution (the pHs studied are 4, 5, 6 and 10.5).

These different pHs make it possible to scan the different ionization states of the carboxyl function of the compounds that they are located below, near and above the pKa (average pKa of Glc-AHESU = 4.88; average pKa of Glc -AHUD = 5, determined by acid-base assay).

Oily phase

Dodecane is used as a model oil.

Emulsification

The oil is added slowly to the aqueous solution while homogenizing using an Ultra Turrax T25 (Janke & Kunkel, IKA) set at 3000 rpm.

The final percentage of oil is 20% by weight.

The preparation is then stirred for 10 minutes by increasing the speed of the Ultra Turrax to 9,000 rpm.

The kinetic evolution of the emulsions thus formed is followed over time by laser granulometry (static light scattering) (Mastersizer 2000, Malvern Instruments), optical microscope and macroscopic observation.

The particle size distribution of the emulsions is characterized in terms of the volume average drop diameter, D(4.3), and polydispersity, P, defined above ([Chem 1] and [Chem 2]). 8.2. Emulsions obtained with Glc-AHESU

8.2.1. Characterization of the emulsions obtained with Glc-AHESU

Figure 15 shows the characteristics of the emulsions on the day of their preparation (JO) at the different pHs studied.

The emulsions formed have a milky white appearance. The initial particle sizes are generally quite similar whatever the pH: the average diameter is of the order of 4-5 pm and the polydispersity is between 0.27 and 0.49. The emulsion at pH=10.5 is more polydisperse than the others with an enlargement towards small diameters.

The emulsion at pH=4 destabilizes by coalescence (fusion between drops) in the hours following its manufacture and is destroyed after 3 days (macroscopic separation of the oil and the aqueous phase) (Figure 16). Emulsions at pH=5, 6 and 10.5 are kinetically stable over time for at least 6 months. The size of the drops remains almost unchanged as shown by monitoring the diameter of the drops D(4.3) as a function of time (Figure 16).

We can also note a creaming phenomenon (Figure 16), which consists of the concentration of oil drops on the surface of the emulsion under the effect of gravity, the drops having a density lower than that of the aqueous phase. .

The glucosylation product Glc-AHESU therefore makes it possible to stabilize emulsions whose aqueous phase has a pH greater than the pKa. The instability observed at low pH reflects the existence of a strong electrostatic component in the stabilization of emulsions. The electrostatic repulsion that prevents droplet coalescence (recombination) results from charged surfactant molecules adsorbed at interfaces.

8.2.2 Study of the destabilization of an emulsion obtained with Glc-AHESU by pH modulation

Due to the presence of an acid function on Glc-AHESU and the pH dependence of the stability of the emulsions described above (coalescence observed quickly at pH=4), we can envisage obtaining pH-stimulable emulsions, ie stable in a range of pH and destabilizing at a given pH, “on demand”.

Tests have been carried out for this purpose (Figure 17).

The initial emulsion is prepared at pH=6, pH where the emulsion is stable over time as shown previously. An HCI solution (0.1 M) is then added to drop the pH to 2. The emulsion is completely destroyed after 24 hours.

The emulsions can also be stimulated by adding salt. Indeed, an emulsion at pH 10.5 prepared with 0.1 M NaCl destabilizes after five days while it is still stable after 6 months in the absence of salt. This supports the hypothesis according to which the stabilization is of electrostatic origin to the extent that it is known that the addition of salt screens electrostatic repulsion.

8.3. Properties of emulsions obtained with Glc-AHUD

8.3.1. Characterization of emulsions obtained with Glc-AHUD

Figure 18 shows the characteristics of the emulsions on the day of their preparation (JO) at the different pHs studied.

The emulsions formed have a milky white appearance. The initial particle sizes are generally quite similar for pHs greater than 5: the average diameter is of the order of 4-5 pm and the polydispersity is between 0.30 and 0.34. Note that at pH=4 the average diameter of the drops is larger (8.51 pm), with a polydispersity of 0.39.

The emulsion at pH = 3.6 is destroyed after 1 day (Figure 19). Emulsions at pH=5; 6.1 and 10.5 are kinetically stable over time for at least 14 days.

The size of the drops remains almost unchanged for up to 14 days as shown by monitoring the diameter of the drops D(4.3) as a function of time (Figure 19). We observe on the emulsions at pH 5 and pH 6.1 the appearance of a thin layer of oil between 14 and 30 days, reflecting a destabilization of the emulsion by coalescence. The particle size measurements are interrupted as soon as this layer visually becomes perceptible. For pH 10.5, a progressive increase in the size of the drops without the macroscopic appearance being impacted at this stage is observed. The 6-month follow-up shows the appearance of a thin layer of oil. For all emulsions, we note a creaming phenomenon which consists of the concentration of oil drops on the surface of the emulsion under the effect of gravity, the drops having a density lower than that of the aqueous phase. This phenomenon can be limited by adding a thickener to the aqueous phase.

Glc-AHUD therefore acts as a surfactant stabilizing emulsions at pH values higher than pKa and more particularly at strongly basic pH (greater stability at pH 10.5 than at pH 6). The instability observed at low pH reflects the existence of a strong electrostatic component in the stabilization of emulsions. The electrostatic repulsion that prevents droplet coalescence results from charged surfactant molecules adsorbed at interfaces.

8.3.2. Study of the destabilization of an emulsion obtained with Glc-AHUD by pH modulation

Glc-AHU makes it possible to manufacture pH-stimulable emulsions, that is to say stable in a range of pH and which destabilize at a given pH, “on demand”.

Preliminary tests were carried out for this purpose (Figure 20). The initial emulsion is prepared at pH 6, pH where the emulsion is stable over time as shown previously. An HCI solution (0.1 M) is then added to drop the pH to 2. The emulsion is completely destabilized after two hours.

8.4. Self-motility properties of emulsions prepared with Glc-AHESU and Glc-AHUD

An auto-motility phenomenon was observed with an emulsion prepared with Glc-AHESU and Glc-AHUD.

Indeed, when a drop of emulsion (approximately 20 pL) is deposited on a solid substrate (glass slide, plastic support, support modified in order to modify the hydrophobicity), we observe that it is subjected to very intense convective phenomena. Drop not only moves but spreads and contracts spasmodically and periodically on its support.

A phenomenon consisting of the sudden appearance/disappearance of transparent zones near the surface of the emulsion was also observed with the emulsions prepared with Glc-AHESU and Glc-AHUD.

Indeed, in certain cases, transparent areas (non-milky) and therefore depleted of drops, form spontaneously near the surface of the drop and disappear immediately (a phenomenon similar to a boiling phenomenon without, however, only bubbles). are not formed). Here again, the phenomenon seems to occur with a certain periodicity of the order of a few seconds to a few minutes depending on the composition of the systems.

These phenomena depend on numerous factors: evaporation, nature of the support, size of the dispersed phase drops and concentration, nature of the oil as shown in Figure 21 which summarizes the influence of these parameters on the intensity of these phenomena.

These phenomena are likely the manifestation of the Marangoni effect, in different forms (R. T. van Gaalen, C. Diddens, H.M.A. Wijshoff, J. G. M. Kuerten (2021). Marangoni circulation in evaporating droplets in the presence of soluble surfactants, Journal of Colloid and Interface Science, Volume 584, Pages 622-633). This results in the convection of matter on a large scale, following the appearance of an interfacial tension gradient.

Such emulsions have the advantage of being “self-spreading” and “self-stirring”, which makes it possible to envisage different applications in the field of surface cleaning.

Example 9: Study of the preparation of dry emulsions using glycolipids according to the invention

According to another embodiment, the composition is in the form of a dry powder, said dry powder being advantageously redispersible in water. The redispersible dry form has many advantages: ease of transport, extended shelf life due to the absence of bacteriological development. Generally, emulsions stabilized by surfactants destabilize during drying (oil exudation). Advantageously, the emulsions based on the glycolipids synthesized in the present invention can be dried without destabilizing and are redispersible in water.

9.1. Material and methods

9.1.1. Preparation of a primary emulsion

Aqueous phase

An aqueous solution comprising 2% by weight of Glc-AHESU and 10% lactose is prepared. Lactose is added to the aqueous phase as a cryoprotective agent.

The pH of the aqueous solution is adjusted to pH=6 using a NaOH solution.

Oily phase

Dodecane is used as a model oil.

Emulsification

The oil is added slowly to the aqueous solution while homogenizing using an Ultra Turrax T25 (Janke & Kunkel, I KA) set at 3000 rpm.

The oil percentage is 20% by weight.

The preparation is then stirred for 10 minutes by increasing the speed of the Ultra Turrax to 9,500 rpm.

A primary emulsion is obtained. Its average diameter is 4.94 pm (P = 0.27) (Figure 22). 9.2.2. Preparation of the dry emulsion

A dry emulsion is obtained by freeze-drying the previously frozen primary emulsion at -80°C.

9.2.3. Redispersion of the dry emulsion

The dry emulsion is redispersed by adding pure water in a quantity equivalent to that evaporated during drying.

9.3. Results

The results show that a primary emulsion prepared with Glc-AHESU can be formed into a dry emulsion and redispersed with gentle stirring. No exudation of oil is observed after drying and the average diameter of the redispersed emulsion is less than 10 pm (Figure 22)

Example 10: Study of the antimicrobial properties of glucosylation products

10.1. Material and methods

Strains of E. Coli K12 are cultured in the presence of concentration of 0% w/v, 0.5% w/v, 1% w/v, 2% w/v and 2.5% w/v of glucosylation products of AHUD or AEA in aqueous phase.

The absorbance of the cultures in the presence of the glucosylation products of AHUD or AEA is monitored at an optical density of 600 nm.

10.2. Results

The results (Figure 23) show that the glucosylation products of AHUD and AEA are bacteriostatic for contents of 1% w/v.

Claims

1. Process for preparing at least one glycolipid corresponding to formula (I): [Glc]n-xOy-R (I) in which

R represents a fatty acid radical comprising between 4 and 24 carbon atoms, the carbon chain of which is linear or branched, saturated or unsaturated, optionally interrupted by one or more sulfur atoms, optionally still being able to comprise one or more chosen substituent(s) (s) among the hydroxyl group, the carbonyl group, the methoxyl group, the amine group, the nitrosyl group, and the thiol group, -xOy- symbolizes the attachment of the fatty acid radical to the saccharide unit by an ether bond connecting an atom of carbon Cx of a glucosyl residue of the osidic unit, previously carrying a hydroxyl group, to a carbon atom Gy of the fatty acid radical, previously carrying a hydroxyl group, with Cx the position of the atom of the glucosyl residue on which is carried out the bond, Cx representing the C1 carbon atom of the glucosyl residue, Cy being a carbon atom positioned along the carbon chain of the fatty acid radical or at the omega end thereof; said process comprising a step of glucosylation of a hydroxylated fatty acid of formula (II):

R-yOH (ll) in which R is a fatty acid radical as defined in formula (I), -yOH represents a hydroxyl group attached to a Cy carbon atom as defined above; said step comprising bringing the hydroxylated fatty acid of formula (II) into contact with at least one a-transglucosylase of the GH70 family in the presence of sucrose or a sucrose analogue.

2. Method according to claim 1, in which the a-transglucosylase of the GH70 family is a branching saccharase of the GH70 family, a glucan-sucrase of the GH70 family, or a mixture of these.

3. Method according to any one of the preceding claims, in which the branching saccharase of the GH70 family for amino acid sequence a sequence chosen from the group comprising GBD-CD2 AN123 (SEQ ID NO: 1), BRS-A (SEQ ID NO: 2), BRS-B A1 (SEQ ID NO: 3), BRS-C (SEQ ID NO: 4), BRS-D A1 (SEQ ID NO: 5), BRS-E A1 (SEQ ID NO: 6), or an amino acid sequence having at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% sequence homology with at least one of the sequences SEQ ID NO: 1 to SEQ ID NO: 6.

4. Method according to claim 3, wherein the branching saccharase of the GH70 family for amino acid sequence a sequence chosen from the group comprising GBD-CD2 AN123 W2135L F2136L (SEQ ID NO: 7), GBD-CD2 AN123 W2135L (SEQ ID NO: 8), GBD-CD2 AN123 W2135I F2136Y (SEQ ID NO: 9), GBD-CD2 AN123 W2135I F2136C (SEQ ID NO: 10), GBD-CD2 AN123 W2135V (SEQ ID NO: 11)), or an amino acid sequence having at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology with at least one of the sequences SEQ ID NO: 7 to SEQ ID NO: 11.

5. Method according to any one of the preceding claims, in which step i) is preferably carried out with a molar ratio of sucrose or sucrose analogue: hydroxylated fatty acid of formula (II) of between 1 and 100, preferably between 10 and 100.

6. Glycolipid corresponding to formula (I): [Glc]n-xOy-R (I) in which [Glc]n represents a linear or branched saccharide unit comprising n glucosyl units, with n between 1 and 7, with the provided that when the saccharide unit comprises several glucosyl units, these are linked together by type a saccharide bonds; R represents a fatty acid radical comprising between 4 and 24 carbon atoms, the carbon chain of which is linear or branched, saturated or unsaturated, optionally interrupted by one or more sulfur atoms, optionally still being able to comprise one or more substituents chosen from the group hydroxyl, the carbonyl group, the methoxyl group, the amine group, the nitrosyl group, and the thiol group, -xOy- symbolizes the attachment of the fatty acid radical to the osidic unit by an ether bond connecting a Cx carbon atom of a residue glucosyl of the saccharide unit, previously carrying a hydroxyl group, to a carbon atom Cy of the fatty acid radical, previously carrying a hydroxyl group, with Cx the position of the atom of the glucosyl residue on which the bond takes place, Cx representing the the C1 carbon atom of the glucosyl residue, Cy being a carbon atom positioned along the carbon chain of the fatty acid radical or at the omega end thereof.

7. Glycolipid according to claim 6, in which the Cy carbon atom is a carbon atom positioned at the omega end of the fatty acid radical.

8. Glycolipid according to any one of claims 6 or 7, in which the glycolipid corresponds to formula (la):

[Glc] _n -xOy-(CH ₂ )bS-(CH ₂ )a-COOH in which a is between 9 and 14, b is greater than or equal to 2, and the sum of a and b is less than 23.

9. Glycolipid according to any one of claims 6 or 7 in which the glycolipid corresponds to the formula (Ib):

10. Glycolipid according to claim 6, in which the fatty acid radical is substituted by one or two hydroxyl groups, and preferably in which: the carbon atom Cy is a carbon atom positioned at the omega end, and the chain carbon of the fatty acid radical is substituted by one or two hydroxyl groups positioned along the carbon chain, or the carbon atom Cy is a carbon atom positioned along the carbon chain, and the carbon chain of the fatty acid radical is substituted by two hydroxyl groups, with one hydroxyl group positioned along the carbon chain and one hydroxyl group positioned in the omega position thereof, or with two hydroxyl groups positioned along the carbon chain.

11. Use of a glycolipid of formula (I) as defined in any one of claims 6 to 10, as an antimicrobial agent.

12. Use of a glycolipid of formula (I) as defined in any one of claims 6 to 10, as a surfactant.

13. Oil-in-water emulsion comprising an oily phase dispersed in an aqueous phase and at least one glycolipid of formula (I) as defined in any one of claims 6 to 10, characterized in that the emulsion is kinetically stable when the pH of the aqueous phase is greater than a threshold hydrogen potential pHs advantageously between 2 and 5.

14. Emulsion according to claim 13 in the form of a dry emulsion.

15. Use of an emulsion according to claim 14 for cleaning surfaces.